Broadly-Neutralizing ANTI-HIV Antibodies

ABSTRACT

The present invention relates to anti-HIV antibodies. Also disclosed are related methods and compositions. HIV causes acquired immunodeficiency syndrome (AIDS), a condition in humans characterized by clinical features including wasting syndromes, central nervous system degeneration and profound immunosuppression that results in life-threatening opportunistic infections and malignancies. Since its discovery in 1981, HIV type 1 (HIV-1) has led to the death of at least 25 million people worldwide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/436,608, filed Apr. 17, 2015, which is the U.S. National Phase ofInternational application Ser. No. PCT/US2013/065696, filed Oct. 18,2013, which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 61/715,642 filed on Oct. 18, 2012, which are herebyincorporated by reference in their entireties.

GOVERNMENT INTERESTS

The invention disclosed herein was made, at least in part, withgovernment support under Grant No. P01 AI081677 from the NationalInstitutes of Health. Accordingly, the U.S. Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates to broad and potent antibodies against HumanImmunodeficiency Virus (“HIV”).

BACKGROUND OF THE INVENTION

HIV causes acquired immunodeficiency syndrome (AIDS), a condition inhumans characterized by clinical features including wasting syndromes,central nervous system degeneration and profound immunosuppression thatresults in life-threatening opportunistic infections and malignancies.Since its discovery in 1981, HIV type 1 (HIV-1) has led to the death ofat least 25 million people worldwide. It is predicted that 20-60 millionpeople will become infected over the next two decades even if there is a2.5% annual decrease in HIV infections. There is a need for therapeuticagents and methods for treatment or inhibition of HIV infection.

Some HIV infected individuals show broadly neutralizing IgG antibodiesin their serum. Yet, little is known regarding the specificity andactivity of these antibodies, despite their potential importance indesigning effective vaccines. In animal models, passive transfer ofneutralizing antibodies can contribute to protection against viruschallenge. Neutralizing antibody responses also can be developed inHIV-infected individuals but the detailed composition of the serologicresponse is yet to be fully uncovered.

SUMMARY OF INVENTION

This invention relates to new categories of broadly-neutralizinganti-HIV antibodies. The consensus heavy and light chain amino acidsequences of the antibodies are listed below and shown in FIGS. 3a and 3b:

(SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLICSVSGX₁SX₂X₃DX₄YWSWIRQSPGKGLEWIGYVHDSGDTNYNPSLKSRVX₅X₆SLDTSKNQVSLKLX₇X₈VTAADSAX₉YYCARAX₁₀HGX₁₁RIYGIVAFGEX₁₂FTYFYMDVWGKGTIVIVSS (SEQ ID NO: 2)SX₁VIRPQPPSLSVAPGETARIX₂CGEX₃SLGSRAVQWYQQRPGQAPSLIIYNNQDRPSGIPERFSGSPDX₄X₅FGTTATLTITX₆VEAGDEADYYCHIW DSRX₇PTX₈WVFGGGITLTVL

In the sequence of SEQ ID NO: 1 or 2, each “X” can be any amino acidresidue or no amino acid. Preferably, each of the Xs can be a residue atthe corresponding location of clonal variants 10-259, 10-303, 10-410,10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, and 10-1369as shown in FIGS. 3a and 3b , and an artificially modified version of10-1074 antibody, 10-1074GM.

Accordingly, one aspect of this invention features an isolated anti-HIVantibody, or antigen binding portion thereof, having at least onecomplementarity determining region (CDR) having a sequence selected fromthe group consisting of SEQ ID NOs: 33-38, with a proviso that theantibody is not antibody PGT-121, 122, or 123. SEQ ID NOs: 33-38 referto the sequences of heavy chain CDRs (CDRH) 1-3 and the light chain CDRs(CDRL) 1-3 under the Kabat system as shown in FIGS. 3a and 3b . In oneembodiment, the CDR can contain a sequence selected from the groupconsisting of SEQ ID NOs: 39-104, i.e., the CDR sequences under theKABAT system as shown in Table 1 below. Alternatively, the CDR cancontain a sequence selected from those corresponding antibodies' CDRsequences under the IMGT system as shown in Table 1 below.

In one embodiment, the isolated anti-HIV antibody, or antigen bindingportion thereof, contains a heavy chain variable region that includesCDRH 1, CDRH 2, and CDRH 3, wherein the CDRH 1, CDRH 2 and CDRH 3include the respective sequences of SEQ ID NOs: 33-35. The CDRH 1, CDRH2 and CDRH 3 can also include the respective sequences of a CDRH setselected from the group consisting of SEQ ID NOs: 39-41, SEQ ID NOs:45-47, SEQ ID NOs: 51-53, SEQ ID NOs: 57-59, SEQ ID NOs: 63-65, SEQ IDNOs: 69-71, SEQ ID NOs: 75-77, SEQ ID NOs: 81-83, SEQ ID NOs: 87-89, SEQID NOs: 93-95, SEQ ID NOs: 99-101, and SEQ ID NOs: 131-133..Alternatively, the CDRHs can contain the respective sequences selectedfrom those corresponding antibodies' CDR sequences under the IMGT systemas shown in Table 1 below.

In another embodiment, the isolated anti-HIV antibody, or antigenbinding portion thereof, contains a light chain variable region thatincludes CDRL 1, CDRL 2 and CDRL 3, wherein the CDRL 1, CDRL 2 and CDRL3 include the respective sequences of SEQ ID NOs: 36-38. For example,the CDRL 1, CDRL 2 and CDRL 3 can include the respective sequences of aCDRL set selected from the group consisting of SEQ ID NOs: 42-44, SEQ IDNOs: 48-50, SEQ ID NOs: 54-56, SEQ ID NOs: 60-62, SEQ ID NOs: 66-68, SEQID NOs: 72-74, SEQ ID NOs: 78-80, SEQ ID NOs: 84-86, SEQ ID NOs: 90-92,SEQ ID NOs: 96-98, SEQ ID NOs: 102-104, and SEQ ID NOs: 134-136.Alternatively, the CDRLs can contain the respective sequences selectedfrom those corresponding antibodies' CDR sequences under the IMGT systemas shown in Table 1 below.

In yet another embodiment, the above-mentioned isolated anti-HIVantibody, or antigen binding portion thereof, includes (i) a heavy chainvariable region that include CDRH 1, CDRH 2, and CDRH 3, and (ii) alight chain variable region that include CDRL 1, CDRL 2 and CDRL 3. TheCDRH 1, CDRH 2, CDRH 3, CDRL 1, CDRL 2 and CDRL 3 can include therespective sequences of a CDR set selected from the group consisting ofSEQ ID NOs: 39-44, SEQ ID NOs: 45-50, SEQ ID NOs: 51-56, SEQ ID NOs:57-62, SEQ ID NOs: 63-68, SEQ ID NOs: 69- 74, SEQ ID NOs: 75-79, SEQ IDNOs: 81-86, SEQ ID NOs: 87-92, SEQ ID NOs: 93-98, SEQ ID NOs: 99-104,and SEQ ID NOs: 131-136. Alternatively, the CDRHs and CDRLs can containthe respective sequences selected from those corresponding antibodies'CDR sequences under the IMGT system as shown in Table 1 below.

In a further embodiment, the isolated anti-HIV antibody, or antigenbinding portion thereof, contains one or both of (i) a heavy chainhaving the consensus amino acid sequence of SEQ ID NO: 1 and (ii) alight chain having the consensus amino acid sequence of SEQ ID NO: 2.The heavy chain can contain a sequence selected from the groupconsisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and129, and the light chain can contain a sequence selected from the groupconsisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and130. For example, the heavy chain and the light chain can include therespective sequences of SEQ ID NOs: 3-4, SEQ ID NOs: 5-6, SEQ ID NOs:7-8, SEQ ID NOs: 9-10, SEQ ID NOs: 11-12, SEQ ID NOs: 13-14, SEQ ID NOs:15-16, SEQ ID NOs: 17-18, SEQ ID NOs: 19-20, SEQ ID NOs: 21-22, SEQ IDNOs: 23-24, and 129-130.

In a preferred embodiment, the isolated anti-HIV antibody is oneselected from the group consisting of 10-259, 10-303, 10-410, 10-847,10-996, 10-1074, 10-1074GM, 10-1121, 10-1130, 10-1146, 10-1341, and10-1369. Their corresponding heavy chain variable regions, light chainvariable regions, CDRH 1-3 and CDRL 1-3 are shown in FIGS. 3a and 3b .In a more preferred embodiment, the isolated anti-HIV antibody is a10-1074-like antibody, i.e., one reselected from the group consisting of10-847, 10-996, 10-1074, 10-1074GM, 10-1146, and 10-1341. An antibody ofthis group is more potent in neutralizing contemporary viruses thanPGT121. The above-discussed antibody can be a human antibody, ahumanized antibody, or a chimeric antibody.

In a second aspect, the invention provides an isolated nucleic acidhaving a sequence encoding a CDR, a heavy chain variable region, or alight chain variable region of the above-discussed anti-HIV antibody, orantigen binding portion thereof. Also featured are a vector having thenucleic acid and a cultured cell having the vector.

The nucleic acid, vector, and cultured cell can be used in a method formaking an anti-HIV antibody or a fragment thereof. The method includes,among others, the steps of: obtaining the cultured cell mentioned above;culturing the cell in a medium under conditions permitting expression ofa polypeptide encoded by the vector and assembling of an antibody orfragment thereof, and purifying the antibody or fragment from thecultured cell or the medium of the cell.

In a third aspect, the invention features a pharmaceutical compositioncontaining (i) at least one anti-HIV antibody mentioned above, orantigen binding portion thereof, and (ii) a pharmaceutically acceptablecarrier.

In a fourth aspect, the invention provides a method of preventing ortreating an HIV infection or an HIV-related disease. The methodincludes, among others, the steps of: identifying a patient in need ofsuch prevention or treatment, and administering to said patient a firsttherapeutic agent containing a therapeutically effective amount of atleast one anti-HIV antibody mentioned above, or antigen binding portionthereof. The method can further include administering a secondtherapeutic agent, such as an antiviral agent.

In a fifth aspect, the invention provides a kit having apharmaceutically acceptable dose unit of a pharmaceutically effectiveamount of at least one isolated anti-HIV antibody mentioned a above, orantigen binding portion thereof, and a pharmaceutically acceptable doseunit of a pharmaceutically effective amount of an anti-HIV agent. Thetwo pharmaceutically acceptable dose units can optionally take the formof a single pharmaceutically acceptable dose unit. Exemplary anti-HIVagent can be one selected from the group consisting of a non-nucleosidereverse transcriptase inhibitor, a protease inhibitor, a entry or fusioninhibitor, and an integrase inhibitor.

In a sixth aspect, the invention provides a kit for the diagnosis,prognosis or monitoring the treatment of an HIV infection in a subject.The kit contains one or more detection reagents which specifically bindto anti-HIV neutralizing antibodies in a biological sample from asubject. The kit can further include reagents for performing PCR or massspectrometry.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D and 1E show: Neutralization activity of PGT121-likeand 10-1074-like variants. (A) Heat map comparing the neutralizationpotencies of PGT121-like and 10-1074-like antibodies in the TZM-b1assay. Darker colors=more potent neutralization; white=noneutralization. (B) Correlation between the mean IC₈₀ against 9 viruses(y axis) and apparent K_(D) values for binding to gp120 and gp140 (xaxis). (C) Graph comparing the neutralization breadth and potencies ofPGT121, 10-996 and 10-1074 antibodies in the TZM-b1 assay against anextended panel of 119 viruses. The y axis shows the cumulative frequencyof IC₅₀ so values up to the concentration shown on the x axis. Thespider graph (upper left corner) shows the frequency distribution ofneutralized viruses according to HIV-1 clades. (D) Dot plot showingmolar neutralization ratios (MNRs; ratio of the Fab and IgG IC₅₀concentrations). Horizontal bars represent the mean ICs₅₀s for allviruses. (E) Bar graph comparing the neutralization potencies of PGT121(dark gray) and 10-1074 (light gray) against viruses isolated fromhistorical (Hist.) and contemporary (Cont.) seroconverters. ns, nonsignificant; **, p<0.005. Fold difference between median IC5os for theneutralization of contemporary viruses by PGT121 and 10-1074 isindicated.

FIGS. 2A, 2B and 2C show: Binding and neutralization activities ofPGT121_(GM) and 10-1074_(GM) mutant antibodies. (A) Bar graphs comparingapparent KD values for the binding of 10-1074, PGT121, PGT121_(GM) and10-1074_(GM) antibodies to gp120 and gp140. Error bars indicate the SEMof K_(D) values obtained from three independent experiments. Folddifferences between KD values of “wildtype” vs “glycomutant” antibodiesare indicated. (B) Bar graphs comparing binding of glycans (FIG. 7A) byPGT121 and 10-1074 with mutant antibodies (PGT121_(GM) and10-1074_(GM)). Numerical scores of binding are measured as fluorescenceintensity (means at duplicate spots) for probes arrayed at 5 fmol perspot. (C) Coverage graph comparing the neutralization breadth andpotencies of PGT121, PGT121_(GM), 10-1074 and 10-1074_(GM) antibodies inthe TZM-b1 assay against a panel of 40 viruses.

FIGS. 3A and 3B depict: Sequence alignments of PGT121 and 10-1074 clonalvariants. (A) Amino acid alignment of the heavy chains (IgH) of thePGT121-like and 10-1074-like antibodies, and the likely germline (GL) VHfor all clonal variants. Amino acid numbering based on crystalstructures, framework (FWR) and complementary determining regions (CDR)as defined by Kabat (. J Exp Med 132(2):211-250) and IMGT (Nucleic AcidsRes 37 (Database issue): D1006-1012) are indicated. Color shading showsacidic (red), basic (blue), and tyrosine (green) amino acids. (B) Sameas A but for the light chains (IgL). FIG. 3A discloses SEQ ID NOS 31, 9,21, 13, 19, 11, 23, 27, 3, 29, 17, 15, 7, 25, 5, and 1, respectively, inorder of appearance. FIG. 3B discloses SEQ ID NOS 32, 26, 6, 24, 30, 4,10, 14, 22, 12, 20, 8, 28, 18, 16, and 2, respectively, in order ofappearance.

FIGS. 4A, 4B and 4C show: Binding affinity of PGT121 and 10-1074 clonalvariants. (A) Binding affinity of the interaction of PGT121 IgG antibodyvariants with YU-2 gp140 and gp120 ligands as measured by surfaceplasmon resonance (SPR). M, mol/l; s, seconds; RU, response units; /, nobinding detected. A chi² value (χ²) <10 indicates that the 1:1 bindingmodel used to fit the curves adequately described the experimental data.Equilibrium and kinetic constants shown are considered as “apparent”constants to account for avidity effects resulting from bivalent bindingof IgGs. (B) Dot plots showing the association (k_(a)) and dissociation(k_(d)) rate constants for PGT121-like (blue shading) and 10-1074-like(green shading). (C) Linear regression graphs comparing the k_(a) andk_(d) values of the IgG antibodies for their binding to gp120 and gp140(x axis) vs their neutralization potencies (mean IC₈₀ values) againstthe 9 viruses shown in Table 4 (y axis).

FIGS. 5A, 5B and 5C depict: Binding of PGT121 variants to gp120 “core”proteins, gp120^(GD324-5AA) mutant and linear gp120^(V3) peptides. (A)ELISA-based binding analyses of PGT121-like and 10-1074-like antibodiesto HXB2 gp120^(core) and 2CC-core proteins compared to intact YU-2gp120. The x axis shows the antibody concentration (M) required toobtain the ELISA values (OD₄₀₅ nm) indicated on the y axis. Theanti-CD4bs antibody VRC01 (Science 329(5993):856-861), the anti-V3 loopantibody 10-188 (PLoS One 6(9): e24078), and the non HIV-reactiveantibody mGO53 (Science 301(5638):1374-1377) were used as controls. (B)Same as (A) but for binding to gp120^(GD324-5AA) mutant protein (c) Bargraphs comparing the ELISA reactivities of the PGT121- and 10-1074-likeantibodies and control antibodies (positive control, 10-188, 1-79, 2-59and 2-1261 (Nature 458(7238):636-640)), and negative control, mGO53)against gp120^(V3-C3) overlapping peptides. The y axis indicates theELISA values (OD₄₀₅ nm) obtained by testing the IgG antibodies at 2μg/ml. The amino acid sequences of individual peptides are shown in thebottom right. All experiments were performed at least in duplicate.Representative data are shown. FIG. 5C discloses SEQ ID NOS 216-223,respectively, in order of appearance.

FIGS. 6A, 6B, 6C and 6D depict: Binding of PGT121 to gp120 glycosylationmutants and deglycosylated gp120. (A) ELISA-based binding analyses ofPGT121 and 10-1074 antibody variants to gp120, gp120^(NNT301-303AAA),gp120^(N332A) and gp120 ^(N332A/NNT301-303AAA). The x axis shows theantibody concentration (M) required to obtain the ELISA values (OD₄₀₅nm) indicated on the y axis. The black dashed and continuous lines showthe averaged reactivity against the four antigens of positive (10-188)and negative (mGO53) antibody controls. (B) Silver-stained SDS-PAGE gelcomparing untreated gp120 (WT, wild type), PNGase F- and EndoH-digestedgp120s. L, protein ladder. (C), Same as (A) but comparing untreated andPNGase F-treated gp120. (D) Same as (A) but comparing untreated andEndoH-treated gp120. All experiments were performed at least induplicate.

FIGS. 7A and 7B depict: Binding of PGT121 and 10-1074 clonal variants toglycans. (FIG. 7A) Monosaccharide sequences of the set of 15 N-glycanprobes used in the glycan microarray analyses to examine PGT121-like and10-1074-like antibodies for direct binding to N-glycans. DH, designatesthe lipid tag 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) towhich the N-glycans were conjugated by reductive amination. Key featuresof note are (i) PGT121-group antibodies bound the monoantennary N-glycanprobe 10 (N2) with a galactose-terminating antenna joined by 1-3-linkageto the core mannose, but not the isomeric N-glycan probe 11 (designatedN4) with the antenna 1-6-linked to the core mannose; (ii) the presenceof this galactose-terminating 1-6-linked antenna, as in the biantennaryprobe 13 (NA2), was permissive to binding, as was the presence ofα2-6-linked (but not α2-3-linked) sialic acid; (iii) the biantennaryprobe 12 (NGA2), lacking galactose and terminating inN-acetylglucosamine, was not bound. (FIG. 7B) Bar graphs comparingglycan binding by PGT121-like, 10-1074-like, and the germline version(GL) antibodies. 10-188, an anti-V3 loop antibody, was used as negativecontrol. Numerical scores of binding are measured as fluorescenceintensity (means at duplicate spots) for probes arrayed at 2 fmol(white) and 5 fmol per spot (grey).

FIGS. 8A, 8B, 8C and 8D depict: Antibody binding and neutralizationactivity against high-mannose-only gp120 and viruses. (A) Silver-stainedSDS-PAGE gel comparing YU-2 gp120 produced in cells treated withkifunensine (gp120_(kif)) and gp120 produced in untreated cells (WT,wild type). L, protein ladder. (B) ELISA comparison of the binding ofPGT121-like (blue labels) and 10-1074-like (green labels) antibodies toYU-2 gp120 (gp120_(WT)) and gp120_(kif). The x axis shows the antibodyconcentration (M) required to obtain the ELISA values (OD₄₀₅ nm)indicated on the y axis. (C) Neutralization curves for PGT121 evaluatedagainst selected PGT121-sensitive/10-1074-resistant pseudovirusesproduced in presence (Virus_(kif)) or absence (Virus_(WT)) ofkifunensine. The dotted horizontal line indicates 50% neutralization,from which the IC₅₀ value can be derived from the antibody concentrationon the x axis. Experiments were performed in triplicate. Error barsindicate the SD of triplicate measurements. (D) Bar graphs comparing theneutralization activity of selected antibodies against YU-2 and PVO.4pseudoviruses produced in HEK 293S GnTI^(−/−)cells (Virus_(GnT) ^(−/−))or in wild type cells (Virus_(WT)). The y axis shows the mean IC₅₀values (μg/ml) for the neutralization of the viruses shown on the xaxis. Error bars indicate the SEM of IC₅₀ so values obtained from twoindependent experiments.

FIGS. 9A, 9B and 9C show: Neutralization activity of PGT121, 10-996 and10-1074. (A) Graphs comparing the neutralization potencies of PGT121,10-996 and 10-74 against viruses of the indicated HIV-1 clades(determined using the TZM-b1 assay and a panel of 119 pseudoviruses).The x axis shows the antibody concentration (μg/ml) required to achieve50% neutralization (IC₅₀). The y axis shows the cumulative frequency ofIC₅₀ values up to the concentration shown on the x axis. (B) Graphcomparing the neutralization breadth and potencies of PGT121, 10-996 and10-1074 antibodies against the extended panel of 119 viruses asdetermined by the TZM-b1 neutralization assay. The y axis shows thecumulative frequency of IC₈₀ values up to the concentration shown on thex axis. (C) Graphs show neutralization curves of the selected viruses byPGT121 and 10-1074. The dotted horizontal line indicates 50%neutralization, from which the IC₅₀ value can be derived from theantibody concentration on the x-axis. Experiments were performed intriplicate. Error bars indicate the SD of triplicate measurements.

FIG. 10 depicts: Neutralization activity against historical vscontemporary clade B viruses. Dot plots comparing neutralizationpotencies against clade B viruses isolated from historical (Hist.) andcontemporary (Cont.) seroconverters for the selected bNAbs. Horizontalbars represent the median IC₅₀ for all viruses per patient. Differencesbetween groups were evaluated using Mann-Whitney test. ns, notsignificant.

FIGS. 11A and 11B depict: Neutralization of two R5 tropic SHIVs with apanel of 11 broadly acting anti-HIV-1 mAbs. The calculated IC50 valuesfor neutralizing SHIVAD8EO (A) and SHIVDH12-V3AD8 (B).

FIG. 12 depicts: The relationship of the plasma concentrations ofpassively administered neutralizing mAbs to virus acquisition followingchallenge of macaques with two different R5 SHIVs. Filled circlesindicate protected (no acquisition) monkeys; open circles denoteinfected animals.

FIGS. 13A, 13B and 13C depict: Plasma concentration of bNAbs. Theconcentration of mAbs was determined by measuring neutralizationactivity in plasma samples. (A) ID50-values measured in TZM.b1neutralization assay of 10-1074 and 3BNC117 against HIV-1 strains thatare sensitive to one but not the other bNAb (i.e. HIV-1 strain X2088_9(10-1074 sensitive); HIV-1 strain Q769_d22 (3BNC117 sensitive). (B)Neutralizing activity of plasma before antibody administration (preP),but spiked with 0.01, 0.1, 1, 10, and 100 μg/ml of antibodies 10-1074(blue) or 3BNC117 (green). Neutralizing activity reported as plasma ipsotiters (left columns) and converted to antibody concentrations (rightcolumns) based on measured ID50-values in (A). (C) ID50 titers (leftcolumns) and concentrations of bNAbs (right columns) measured in theindicated macaque plasma samples before (Prebleed) and following (Day)bNAb administration.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, on an unexpected discovery ofa new category of broadly neutralizing antibodies (bNAbs) against HIVthat can recognize carbohydrate-dependent epitopes, includingcomplex-type N-glycan, on gp120.

Antibodies are essential for the success of most vaccines, andantibodies against HIV appear to be the only correlate of protection inthe recent RV144 anti-HIV vaccine trial. Some HIV-1 infected patientsdevelop broadly neutralizing serologic activity against the gp160 viralspike 2-4 years after infection, but these antibodies do not generallyprotect infected humans because autologous viruses escape throughmutation. Nevertheless, broadly neutralizing activity puts selectivepressure on the virus and passive transfer of broadly neutralizingantibodies (bNAbs) to macaques protects against SHIV infection. It hastherefore been proposed that vaccines that elicit such antibodies may beprotective against HIV infection in humans.

The development of single cell antibody cloning techniques revealed thatbNAbs target several different epitopes on the HIV-1 gp160 spike. Themost potent HIV-1 bNAbs recognize the CD4 binding site (CD4bs) (Science333(6049):1633-1637; Nature 477(7365):466-470; Science334(6060):1289-1293) and carbohydrate-dependent epitopes associated withthe variable loops (Nature 477(7365):466-470; Science 326(5950):285-289;Science 334(6059):1097-1103; Nature 480(7377):336-343), including theV1/V2 (PG9/PG16) (Science 326(5950):285-289) and V3 loops (PGTs) (Nature477(7365):466-470). Less is known about carbohydrate-dependent epitopesbecause the antibodies studied to date are either unique examples ormembers of small clonal families.

To better understand the neutralizing antibody response to HIV-1 and theepitope targeted by PGT antibodies, we isolated members of a largeclonal family dominating the gp160-specific IgG memory response from theclade A-infected patient who produced PGT121. As disclosed herein,PGT121 antibodies segregate into two groups, a PGT121-like and a10-1074-like group, according to sequence, binding affinity,neutralizing activity and recognition of carbohydrates and the V3 loop.10-1074 and related family members exhibit unusual potentneutralization, including broad reactivity against newly-transmittedviruses. Unlike previously-characterized carbohydrate-dependent bNAbs,PGT121 binds to complex-type, rather than high-mannose, N-glycans inglycan microarray experiments. Crystal structures of PGT121 and 10-1074compared with structures of their germline precursor and a structure ofPGT121 bound to a complex-type N-glycan rationalize their distinctproperties.

In one example, assays were carried out to isolate B-cell clonesencoding PGT121, which is unique among glycan-dependent bNAbs inrecognizing complex-type, rather than high-mannose, N-glycans. ThePGT121 clones segregates into PGT121- and 10-1074-like groupsdistinguished by sequence, binding affinity, carbohydrate recognitionand neutralizing activity. The 10-1074 group exhibit remarkable potencyand breadth despite not binding detectably to protein-free glycans.Crystal structures of un-liganded PGT121, 10-1074, and their germlineprecursor reveal that differential carbohydrate recognition maps to acleft between CDRH2 and CDRH3, which was occupied by a complex-typeN-glycan in a separate PGT121 structure. Swapping glycan contactresidues between PGT121 and 10-1074 confirmed the importance of theseresidues in neutralizing activities. HIV envelopes exhibit varyingproportions of high-mannose- and complex-type N-glycans, thus theseresults, including the first structural characterization of complex-typeN-glycan recognition by anti-HIV bNAbs, are critical for understandinghow antibodies and ultimately vaccines might achieve broad neutralizingactivity.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (for example, bispecificantibodies and polyreactive antibodies), and antibody fragments. Thus,the term “antibody” as used in any context within this specification ismeant to include, but not be limited to, any specific binding member,immunoglobulin class and/or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM,IgA, IgD, IgE and IgM); and biologically relevant fragment or specificbinding member thereof, including but not limited to Fab, F(ab′)2, Fv,and scFv (single chain or related entity). It is understood in the artthat an antibody is a glycoprotein having at least two heavy (H) chainsand two light (L) chains inter-connected by disulfide bonds, or anantigen binding portion thereof. A heavy chain is comprised of a heavychain variable region (VH) and a heavy chain constant region (CH1, CH2and CH3). A light chain is comprised of a light chain variable region(VL) and a light chain constant region (CL). The variable regions ofboth the heavy and light chains comprise framework regions (FWR) andcomplementarity determining regions (CDR). The four FWR regions arerelatively conserved while CDR regions (CDR1, CDR2 and CDR3) representhypervariable regions and are arranged from NH2 terminus to the COOHterminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. Thevariable regions of the heavy and light chains contain a binding domainthat interacts with an antigen while, depending of the isotype, theconstant region(s) may mediate the binding of the immunoglobulin to hosttissues or factors.

Also included in the definition of “antibody” as used herein arechimeric antibodies, humanized antibodies, and recombinant antibodies,human antibodies generated from a transgenic non-human animal, as wellas antibodies selected from libraries using enrichment technologiesavailable to the artisan.

The term “variable” refers to the fact that certain segments of thevariable (V) domains differ extensively in sequence among antibodies.The V domain mediates antigen binding and defines specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the 110-amino acid span of the variableregions. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable regions of nativeheavy and light chains each comprise four FRs, largely adopting a betasheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the beta sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see, for example, Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The term “hypervariable region” as used herein refers to the amino acidresidues of an antibody that are responsible for antigen binding. Thehypervariable region generally comprises amino acid residues from a“complementarity determining region” (“CDR”).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The term “polyclonal antibody” refers to preparationsthat include different antibodies directed against differentdeterminants (“epitopes”).

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with, orhomologous to, corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with, orhomologous to, corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, for example, U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).The described invention provides variable region antigen-bindingsequences derived from human antibodies. Accordingly, chimericantibodies of primary interest herein include antibodies having one ormore human antigen binding sequences (for example, CDRs) and containingone or more sequences derived from a non-human antibody, for example, anFR or C region sequence. In addition, chimeric antibodies includedherein are those comprising a human variable region antigen bindingsequence of one antibody class or subclass and another sequence, forexample, FR or C region sequence, derived from another antibody class orsubclass.

A “humanized antibody” generally is considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues often arereferred to as “import” residues, which typically are taken from an“import” variable region. Humanization may be performed following themethod of Winter and co-workers (see, for example, Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting importhypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (see, for example, U.S. Pat. No. 4,816,567), wheresubstantially less than an intact human variable region has beensubstituted by the corresponding sequence from a non-human species.

An “antibody fragment” comprises a portion of an intact antibody, suchas the antigen binding or variable region of the intact antibody.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see, forexample, U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This fragment contains adimer of one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (three loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable region (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” (“sFv” or “scFv”) are antibody fragments that comprisethe VH and VL antibody domains connected into a single polypeptidechain. The sFv polypeptide can further comprise a polypeptide linkerbetween the VH and VL domains that enables the sFv to form the desiredstructure for antigen binding. For a review of sFv, see, for example,Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments with short linkers (about 5-10 residues)between the VH and VL domains such that inter-chain but not intra-chainpairing of the V domains is achieved, resulting in a bivalent fragment,i.e., fragment having two antigen-binding sites. Bispecific diabodiesare heterodimers of two “crossover” sFv fragments in which the VH and VLdomains of the two antibodies are present on different polypeptidechains. Diabodies are described more fully in, for example, EP 404,097;WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993).

Domain antibodies (dAbs), which can be produced in fully human form, arethe smallest known antigen-binding fragments of antibodies, ranging fromabout 11 kDa to about 15 kDa. DAbs are the robust variable regions ofthe heavy and light chains of immunoglobulins (VH and VL, respectively).They are highly expressed in microbial cell culture, show favorablebiophysical properties including, for example, but not limited to,solubility and temperature stability, and are well suited to selectionand affinity maturation by in vitro selection systems such as, forexample, phage display. DAbs are bioactive as monomers and, owing totheir small size and inherent stability, can be formatted into largermolecules to create drugs with prolonged serum half-lives or otherpharmacological activities. Examples of this technology have beendescribed in, for example, WO9425591 for antibodies derived fromCamelidae heavy chain Ig, as well in US20030130496 describing theisolation of single domain fully human antibodies from phage libraries.

Fv and sFv are the only species with intact combining sites that aredevoid of constant regions. Thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins can beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See, for example, AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment also can be a“linear antibody”, for example, as described in U.S. Pat. No. 5,641,870for example. Such linear antibody fragments can be monospecific orbispecific.

In certain embodiments, antibodies of the described invention arebispecific or multi-specific. Bispecific antibodies are antibodies thathave binding specificities for at least two different epitopes.Exemplary bispecific antibodies can bind to two different epitopes of asingle antigen. Other such antibodies can combine a first antigenbinding site with a binding site for a second antigen. Alternatively, ananti-HIV arm can be combined with an arm that binds to a triggeringmolecule on a leukocyte, such as a T-cell receptor molecule (forexample, CD3), or Fc receptors for IgG (Fc gamma R), such as Fc gamma RI(CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD16), so as to focus andlocalize cellular defense mechanisms to the infected cell. Bispecificantibodies also can be used to localize cytotoxic agents to infectedcells. Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (for example, F(ab′)2 bispecific antibodies). Forexample, WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc gammaRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-Fc gamma RI antibody. For example, a bispecificanti-ErbB2/Fc alpha antibody is reported in WO98/02463; U.S. Pat. No.5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody. See also,for example, Mouquet et al., Polyreactivity Increases The ApparentAffinity Of Anti-HIV Antibodies By Heteroligation. Nature. 467, 591-5(2010), and Mouquet et al., Enhanced HIV-1 neutralization by antibodyheteroligation” Proc Natl Acad Sci USA. 2012 Jan.17; 109(3):875-80.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, for example,Millstein et al., Nature, 305:537-539 (1983)). Similar procedures aredisclosed in, for example, WO 93/08829, Traunecker et al., EMBO J.,10:3655-3659 (1991) and see also Mouquet et al., Enhanced HIV-1neutralization by antibody heteroligation” Proc Natl Acad Sci USA. 2012Jan. 17; 109(3):875-80.

Alternatively, antibody variable regions with the desired bindingspecificities (antibody-antigen combining sites) are fused toimmunoglobulin constant domain sequences. The fusion is with an Ig heavychain constant domain, comprising at least part of the hinge, CH2, andCH3 regions. According to some embodiments, the first heavy-chainconstant region (CH1) containing the site necessary for light chainbonding, is present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host cell. This provides for greaterflexibility in adjusting the mutual proportions of the three polypeptidefragments in embodiments when unequal ratios of the three polypeptidechains used in the construction provide the optimum yield of the desiredbispecific antibody. It is, however, possible to insert the codingsequences for two or all three polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant affect on the yield of the desired chain combination.

Techniques for generating bispecific antibodies from antibody fragmentsalso have been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. For example, Brennanet al., Science, 229: 81 (1985) describe a procedure wherein intactantibodies are proteolytically cleaved to generate F(ab′)2 fragments.These fragments are reduced in the presence of the dithiol complexingagent, sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated thenare converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives then is reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Other modifications of the antibody are contemplated herein. Forexample, the antibody can be linked to one of a variety ofnonproteinaceous polymers, for example, polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. The antibody also can be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed in,for example, Remington's Pharmaceutical Sciences, 16th edition, Oslo,A., Ed., (1980).

Typically, the antibodies of the described invention are producedrecombinantly, using vectors and methods available in the art. Humanantibodies also can be generated by in vitro activated B cells (see, forexample, U.S. Pat. Nos. 5,567,610 and 5,229,275). General methods inmolecular genetics and genetic engineering useful in the presentinvention are described in the current editions of Molecular Cloning: ALaboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor LaboratoryPress), Gene Expression Technology (Methods in Enzymology, Vol. 185,edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guideto Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed.,(1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.),Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I.Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.). Reagents, cloning vectors, and kits for geneticmanipulation are available from commercial vendors such as BioRad,Stratagene, Invitrogen, ClonTech and Sigma-Aldrich Co.

Human antibodies also can be produced in transgenic animals (forexample, mice) that are capable of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge. See, for example, Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993);U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S.Pat. No. 5,545,807; and WO 97/17852. Such animals can be geneticallyengineered to produce human antibodies comprising a polypeptide of thedescribed invention.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, for example, Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E. coli,thus allowing the facile production of large amounts of these fragments.Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)2 fragments (see, for example, Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)2 fragments can be isolated directly from recombinant host cellculture. Fab and F(ab′)2 fragment with increased in vivo half-lifecomprising a salvage receptor binding epitope residues are described inU.S. Pat. No. 5,869,046. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

Other techniques that are known in the art for the selection of antibodyfragments from libraries using enrichment technologies, including butnot limited to phage display, ribosome display (Hanes and Pluckthun,1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou,et al., 1997, Nature Biotechnology 15: 29-34) and/or yeast display(Kieke, et al., 1997, Protein Engineering 10: 1303-1310) may be utilizedas alternatives to previously discussed technologies to select singlechain antibodies. Single-chain antibodies are selected from a library ofsingle chain antibodies produced directly utilizing filamentous phagetechnology. Phage display technology is known in the art (e.g., seetechnology from Cambridge Antibody Technology (CAT)) as disclosed inU.S. Pat. Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793;5,962,255; 6,140,471; 6,225,447; 6,291,650; 6,492,160; 6,521,404;6,544,731; 6,555,313; 6,582,915; 6,593, 081, as well as other U.S.family members, or applications which rely on priority filing GB9206318, filed 24 May 1992; see also Vaughn, et al. 1996, NatureBiotechnology 14: 309-314). Single chain antibodies may also be designedand constructed using available recombinant DNA technology, such as aDNA amplification method (e.g., PCR), or possibly by using a respectivehybridoma cDNA as a template.

Variant antibodies also are included within the scope of the invention.Thus, variants of the sequences recited in the application also areincluded within the scope of the invention. Further variants of theantibody sequences having improved affinity can be obtained usingmethods known in the art and are included within the scope of theinvention. For example, amino acid substitutions can be used to obtainantibodies with further improved affinity. Alternatively, codonoptimization of the nucleotide sequence can be used to improve theefficiency of translation in expression systems for the production ofthe antibody.

Such variant antibody sequences will share 70% or more (i.e., 80%, 85%,90%, 95%, 97%, 98%, 99% or greater) sequence identity with the sequencesrecited in the application. Such sequence identity is calculated withregard to the full length of the reference sequence (i.e., the sequencerecited in the application). Percentage identity, as referred to herein,is as determined using BLAST version 2.1.3 using the default parametersspecified by the NCBI (the National Center for BiotechnologyInformation) [Blosum 62 matrix; gap open penalty=11 and gap extensionpenalty=1]. For example, peptide sequences are provided by thisinvention that comprise at least about 5, 10, 15, 20, 30, 40, 50, 75,100, 150, or more contiguous peptides of one or more of the sequencesdisclosed herein as well as all intermediate lengths there between. Asused herein, the term “intermediate lengths” is meant to describe anylength between the quoted values, such as 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.

The present invention provides for antibodies, either alone or incombination with other antibodies, such as, but not limited to, VRC01,anti-V3 loop, CD4bs, and CD4i antibodies as well as PG9/PG16-likeantibodies, that have broad neutralizing activity in serum.

According to another embodiment, the present invention provides methodsfor the preparation and administration of an HIV antibody compositionthat is suitable for administration to a human or non-human primatepatient having HIV infection, or at risk of HIV infection, in an amountand according to a schedule sufficient to induce a protective immuneresponse against HIV, or reduction of the HIV virus, in a human.

According to another embodiment, the present invention provides avaccine comprising at least one antibody of the invention and apharmaceutically acceptable carrier. According to one embodiment, thevaccine is a vaccine comprising at least one antibody described hereinand a pharmaceutically acceptable carrier. The vaccine can include aplurality of the antibodies having the characteristics described hereinin any combination and can further include antibodies neutralizing toHIV as are known in the art.

It is to be understood that compositions can be a single or acombination of antibodies disclosed herein, which can be the same ordifferent, in order to prophylactically or therapeutically treat theprogression of various subtypes of HIV infection after vaccination. Suchcombinations can be selected according to the desired immunity. When anantibody is administered to an animal or a human, it can be combinedwith one or more pharmaceutically acceptable carriers, excipients oradjuvants as are known to one of ordinary skilled in the art. Thecomposition can further include broadly neutralizing antibodies known inthe art, including but not limited to, VRC01, b12, anti-V3 loop, CD4bs,and CD4i antibodies as well as PG9/PG16-like antibodies.

Further, with respect to determining the effective level in a patientfor treatment of HIV, in particular, suitable animal models areavailable and have been widely implemented for evaluating the in vivoefficacy against HIV of various gene therapy protocols (Sarver et al.(1993b), supra). These models include mice, monkeys and cats. Eventhough these animals are not naturally susceptible to HIV disease,chimeric mice models (for example, SCID, bg/nu/xid, NOD/SCID, SCID-hu,immunocompetent SCID-hu, bone marrow-ablated BALB/c) reconstituted withhuman peripheral blood mononuclear cells (PBMCs), lymph nodes, fetalliver/thymus or other tissues can be infected with lentiviral vector orHIV, and employed as models for HIV pathogenesis. Similarly, the simianimmune deficiency virus (SIV)/monkey model can be employed, as can thefeline immune deficiency virus (FIV)/cat model. The pharmaceuticalcomposition can contain other pharmaceuticals, in conjunction with avector according to the invention, when used to therapeutically treatAIDS. These other pharmaceuticals can be used in their traditionalfashion (i.e., as agents to treat HIV infection).

According to another embodiment, the present invention provides anantibody-based pharmaceutical composition comprising an effective amountof an isolated HIV antibody, or an affinity matured version, whichprovides a prophylactic or therapeutic treatment choice to reduceinfection of the HIV virus. The antibody-based pharmaceuticalcomposition of the present invention may be formulated by any number ofstrategies known in the art (e.g., see McGoff and Scher, 2000, SolutionFormulation of Proteins/Peptides: In McNally, E. J., ed. ProteinFormulation and Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158;Akers and Defilippis, 2000, Peptides and Proteins as ParenteralSolutions. In: Pharmaceutical Formulation Development of Peptides andProteins. Philadelphia, Pa.: Talyor and Francis; pp. 145-177; Akers, etal., 2002, Pharm. Biotechnol. 14:47-127). A pharmaceutically acceptablecomposition suitable for patient administration will contain aneffective amount of the antibody in a formulation which both retainsbiological activity while also promoting maximal stability duringstorage within an acceptable temperature range. The pharmaceuticalcompositions can also include, depending on the formulation desired,pharmaceutically acceptable diluents, pharmaceutically acceptablecarriers and/or pharmaceutically acceptable excipients, or any suchvehicle commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected so as not toaffect the biological activity of the combination. Examples of suchdiluents are distilled water, physiological phosphate-buffered saline,Ringer's solutions, dextrose solution, and Hank's solution. The amountof an excipient that is useful in the pharmaceutical composition orformulation of this invention is an amount that serves to uniformlydistribute the antibody throughout the composition so that it can beuniformly dispersed when it is to be delivered to a subject in needthereof. It may serve to dilute the antibody to a concentration whichprovides the desired beneficial palliative or curative results while atthe same time minimizing any adverse side effects that might occur fromtoo high a concentration. It may also have a preservative effect. Thus,for the antibody having a high physiological activity, more of theexcipient will be employed. On the other hand, for any activeingredient(s) that exhibit a lower physiological activity, a lesserquantity of the excipient will be employed.

The above described antibodies and antibody compositions or vaccinecompositions, comprising at least one or a combination of the antibodiesdescribed herein, can be administered for the prophylactic andtherapeutic treatment of HIV viral infection.

The present invention also relates to isolated polypeptides comprisingthe novel amino acid sequences of the light chains and heavy chains, aswell as the consensus sequences for the heavy and light chains of SEQ IDNOs: 1 and 2, as listed in FIG. 3.

In other related embodiments, the invention provides polypeptidevariants that encode the amino acid sequences of the HIV antibodieslisted in FIG. 3; the consensus sequences for the heavy and light chainsof SEQ ID NOs: 1 and 2. These polypeptide variants have at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or greater, sequenceidentity compared to a polypeptide sequence of this invention, asdetermined using the methods described herein, (for example, BLASTanalysis using standard parameters). One skilled in this art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by taking into amino acidsimilarity and the like.

The term “polypeptide” is used in its conventional meaning, i.e., as asequence of amino acids. The polypeptides are not limited to a specificlength of the product. Peptides, oligopeptides, and proteins areincluded within the definition of polypeptide, and such terms can beused interchangeably herein unless specifically indicated otherwise.This term also includes post-expression modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring. A polypeptide can be anentire protein, or a subsequence thereof. Particular polypeptides ofinterest in the context of this invention are amino acid subsequencescomprising CDRs, VH and VL, being capable of binding an antigen orHIV-infected cell.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants can be naturally occurring or can be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating one or morebiological activities of the polypeptide as described herein and/orusing any of a number of techniques well known in the art.

For example, certain amino acids can be substituted for other aminoacids in a protein structure without appreciable loss of its ability tobind other polypeptides (for example, antigens) or cells. Since it isthe binding capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, accordingly, itsunderlying DNA coding sequence, whereby a protein with like propertiesis obtained. It is thus contemplated that various changes can be made inthe peptide sequences of the disclosed compositions, or correspondingDNA sequences that encode said peptides without appreciable loss oftheir biological utility or activity.

In many instances, a polypeptide variant will contain one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged.

Amino acid substitutions generally are based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

“Homology” or “sequence identity” refers to the percentage of residuesin the polynucleotide or polypeptide sequence variant that are identicalto the non-variant sequence after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent homology. Inparticular embodiments, polynucleotide and polypeptide variants have atleast about 70%, at least about 75%, at least about 80%, at least about90%, at least about 95%, at least about 98%, or at least about 99%polynucleotide or polypeptide homology with a polynucleotide orpolypeptide described herein.

Such variant polypeptide sequences will share 70% or more (i.e. 80%,85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with thesequences recited in the application. In additional embodiments, thedescribed invention provides polypeptide fragments comprising variouslengths of contiguous stretches of amino acid sequences disclosedherein. For example, peptide sequences are provided by this inventionthat comprise at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, ormore contiguous peptides of one or more of the sequences disclosedherein as well as all intermediate lengths there between.

The invention also includes nucleic acid sequences encoding part or allof the light and heavy chains of the described inventive antibodies, andfragments thereof. Due to redundancy of the genetic code, variants ofthese sequences will exist that encode the same amino acid sequences.

The present invention also includes isolated nucleic acid sequencesencoding the polypeptides for the heavy and light chains of the HIVantibodies listed in FIG. 3 and the consensus sequences for the heavyand light chains of SEQ ID NOs: 1 and 2.

In other related embodiments, the described invention providespolynucleotide variants that encode the peptide sequences of the heavyand light chains of the HIV antibodies listed in FIG. 3; the consensussequences for the heavy and light chains of SEQ ID NOs: 1 and 2. Thesepolynucleotide variants have at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, or greater, sequence identity compared to apolynucleotide sequence of this invention, as determined using themethods described herein, (for example, BLAST analysis using standardparameters). One skilled in this art will recognize that these valuescan be appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to single-stranded or double-stranded RNA, DNA, or mixedpolymers. Polynucleotides can include genomic sequences, extra-genomicand plasmid sequences, and smaller engineered gene segments thatexpress, or can be adapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantiallyseparated from other genome DNA sequences as well as proteins orcomplexes such as ribosomes and polymerases, which naturally accompany anative sequence. The term encompasses a nucleic acid sequence that hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analoguesor analogues biologically synthesized by heterologous systems. Asubstantially pure nucleic acid includes isolated forms of the nucleicacid. Accordingly, this refers to the nucleic acid as originallyisolated and does not exclude genes or sequences later added to theisolated nucleic acid by the hand of man.

A polynucleotide “variant,” as the term is used herein, is apolynucleotide that typically differs from a polynucleotide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants can be naturally occurring or can besynthetically generated, for example, by modifying one or more of thepolynucleotide sequences of the invention and evaluating one or morebiological activities of the encoded polypeptide as described hereinand/or using any of a number of techniques well known in the art.

Modifications can be made in the structure of the polynucleotides of thedescribed invention and still obtain a functional molecule that encodesa variant or derivative polypeptide with desirable characteristics. Whenit is desired to alter the amino acid sequence of a polypeptide tocreate an equivalent, or even an improved, variant or portion of apolypeptide of the invention, one skilled in the art typically willchange one or more of the codons of the encoding DNA sequence.

Typically, polynucleotide variants contain one or more substitutions,additions, deletions and/or insertions, such that the immunogenicbinding properties of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein.

In additional embodiments, the described invention providespolynucleotide fragments comprising various lengths of contiguousstretches of sequence identical to or complementary to one or more ofthe sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 10, 15, 20, 30,40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between and encompass any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; and including all integers through 200-500; 500-1,000.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderate stringent conditions for testing thehybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50-60° C., 5×SSC, overnight; followedby washing twice at 65° C. for 20 minutes with each of 2×, 0.5×, and0.2×SSC containing 0.1% SDS. One skilled in the art will understand thatthe stringency of hybridization can be readily manipulated, such as byaltering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, for example, to 60-65° C. or 65-70° C.

In some embodiments, the polypeptide encoded by the polynucleotidevariant or fragment has the same binding specificity (i.e., specificallyor preferentially binds to the same epitope or HIV strain) as thepolypeptide encoded by the native polynucleotide. In some embodiments,the described polynucleotides, polynucleotide variants, fragments andhybridizing sequences, encode polypeptides that have a level of bindingactivity of at least about 50%, at least about 70%, and at least about90% of that for a polypeptide sequence specifically set forth herein.

The polynucleotides of the described invention, or fragments thereof,regardless of the length of the coding sequence itself, can be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length can varyconsiderably. A nucleic acid fragment of almost any length is employed.For example, illustrative polynucleotide segments with total lengths ofabout 10000, about 5000, about 3000, about 2000, about 1000, about 500,about 200, about 100, about 50 base pairs in length, and the like,(including all intermediate lengths) are included in manyimplementations of this invention.

Further included within the scope of the invention are vectors such asexpression vectors, comprising a nucleic acid sequence according to theinvention. Cells transformed with such vectors also are included withinthe scope of the invention.

The present invention also provides vectors and host cells comprising anucleic acid of the invention, as well as recombinant techniques for theproduction of a polypeptide of the invention. Vectors of the inventioninclude those capable of replication in any type of cell or organism,including, for example, plasmids, phage, cosmids, and mini chromosomes.In some embodiments, vectors comprising a polynucleotide of thedescribed invention are vectors suitable for propagation or replicationof the polynucleotide, or vectors suitable for expressing a polypeptideof the described invention. Such vectors are known in the art andcommercially available.

“Vector” includes shuttle and expression vectors. Typically, the plasmidconstruct also will include an origin of replication (for example, theColEl origin of replication) and a selectable marker (for example,ampicillin or tetracycline resistance), for replication and selection,respectively, of the plasmids in bacteria. An “expression vector” refersto a vector that contains the necessary control sequences or regulatoryelements for expression of the antibodies including antibody fragment ofthe invention, in bacterial or eukaryotic cells.

As used herein, the term “cell” can be any cell, including, but notlimited to, that of a eukaryotic, multicellular species (for example, asopposed to a unicellular yeast cell), such as, but not limited to, amammalian cell or a human cell. A cell can be present as a singleentity, or can be part of a larger collection of cells. Such a “largercollection of cells” can comprise, for example, a cell culture (eithermixed or pure), a tissue (for example, endothelial, epithelial, mucosaor other tissue), an organ (for example, lung, liver, muscle and otherorgans), an organ system (for example, circulatory system, respiratorysystem, gastrointestinal system, urinary system, nervous system,integumentary system or other organ system), or an organism (e.g., abird, mammal, or the like).

Polynucleotides of the invention may synthesized, whole or in parts thatthen are combined, and inserted into a vector using routine molecularand cell biology techniques, including, for example, subcloning thepolynucleotide into a linearized vector using appropriate restrictionsites and restriction enzymes. Polynucleotides of the describedinvention are amplified by polymerase chain reaction usingoligonucleotide primers complementary to each strand of thepolynucleotide. These primers also include restriction enzyme cleavagesites to facilitate subcloning into a vector. The replicable vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, and one or moremarker or selectable genes.

In order to express a polypeptide of the invention, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into an appropriate expression vector, i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

The present invention also provides kits useful in performing diagnosticand prognostic assays using the antibodies, polypeptides and nucleicacids of the present invention. Kits of the present invention include asuitable container comprising an HIV antibody, a polypeptide or anucleic acid of the invention in either labeled or unlabeled form. Inaddition, when the antibody, polypeptide or nucleic acid is supplied ina labeled form suitable for an indirect binding assay, the kit furtherincludes reagents for performing the appropriate indirect assay. Forexample, the kit may include one or more suitable containers includingenzyme substrates or derivatizing agents, depending on the nature of thelabel. Control samples and/or instructions may also be included. Thepresent invention also provides kits for detecting the presence of theHIV antibodies or the nucleotide sequence of the HIV antibody of thepresent invention in a biological sample by PCR or mass spectrometry.

“Label” as used herein refers to a detectable compound or compositionthat is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. A label can also be conjugated to apolypeptide and/or a nucleic acid sequence disclosed herein. The labelcan be detectable by itself (for example, radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, can catalyzechemical alteration of a substrate compound or composition that isdetectable. Antibodies and polypeptides of the described invention alsocan be modified to include an epitope tag or label, for example, for usein purification or diagnostic applications. Suitable detection meansinclude the use of labels such as, but not limited to, radionucleotides,enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzymesubstrates or co-factors, enzyme inhibitors, prosthetic group complexes,free radicals, particles, dyes, and the like.

According to another embodiment, the present invention providesdiagnostic methods. Diagnostic methods generally involve contacting abiological sample obtained from a patient, such as, for example, blood,serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy,with an HIV antibody and determining whether the antibody preferentiallybinds to the sample as compared to a control sample or predeterminedcut-off value, thereby indicating the presence of the HIV virus.

According to another embodiment, the present invention provides methodsto detect the presence of the HIV antibodies of the present invention ina biological sample from a patient. Detection methods generally involveobtaining a biological sample from a patient, such as, for example,blood, serum, saliva, urine, sputum, a cell swab sample, or a tissuebiopsy and isolating HIV antibodies or fragments thereof, or the nucleicacids that encode an HIV antibody, and assaying for the presence of anHIV antibody in the biological sample. Also, the present inventionprovides methods to detect the nucleotide sequence of an HIV antibody ina cell. The nucleotide sequence of an HIV antibody may also be detectedusing the primers disclosed herein. The presence of the HIV antibody ina biological sample from a patient may be determined utilizing knownrecombinant techniques and/or the use of a mass spectrometer.

In another embodiment, the present invention provides a method fordetecting an HIV antibody comprising a heavy chain comprising a highlyconserved consensus sequence and a light chain comprising a highlyconserved consensus sequence in a biological sample, comprisingobtaining an immunoglobulin-containing biological sample from amammalian subject, isolating an HIV antibody from said sample, andidentifying the highly conserved consensus sequences of the heavy chainand the light chain. The biological sample may be blood, serum, saliva,urine, sputum, a cell swab sample, or a tissue biopsy. The amino acidsequences may be determined by methods known in the art including, forexample, PCR and mass spectrometry.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the presence of”includes determining the amount of something present, and/or determiningwhether it is present or absent. As used herein, the terms“determining,” “measuring,” and “assessing,” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations.

II. Method of Reducing Viral Replication

Methods for reducing an increase in HIV virus titer, virus replication,virus proliferation or an amount of an HIV viral protein in a subjectare further provided. According to another aspect, a method includesadministering to the subject an amount of an HIV antibody effective toreduce an increase in HIV titer, virus replication or an amount of anHIV protein of one or more HIV strains or isolates in the subject.

According to another embodiment, the present invention provides a methodof reducing viral replication or spread of HIV infection to additionalhost cells or tissues comprising contacting a mammalian cell with theantibody, or a portion thereof, which binds to an antigenic epitope ongp120.

III. Method of Treatment

According to another embodiment, the present invention provides a methodfor treating a mammal infected with a virus infection, such as, forexample, HIV, comprising administering to said mammal a pharmaceuticalcomposition comprising the HIV antibodies disclosed herein. According toone embodiment, the method for treating a mammal infected with HIVcomprises administering to said mammal a pharmaceutical composition thatcomprises an antibody of the present invention, or a fragment thereof.The compositions of the invention can include more than one antibodyhaving the characteristics disclosed (for example, a plurality or poolof antibodies). It also can include other HIV neutralizing antibodies asare known in the art, for example, but not limited to, VRC01, PG9 andb12.

Passive immunization has proven to be an effective and safe strategy forthe prevention and treatment of viral diseases. (See, for example,Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat.Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999);and Igarashi et al., Nat. Med. 5:211-16 (1999). Passive immunizationusing human monoclonal antibodies provides an immediate treatmentstrategy for emergency prophylaxis and treatment of HIV.

Subjects at risk for HIV-related diseases or disorders include patientswho have come into contact with an infected person or who have beenexposed to HIV in some other way. Administration of a prophylactic agentcan occur prior to the manifestation of symptoms characteristic ofHIV-related disease or disorder, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

For in vivo treatment of human and non-human patients, the patient isadministered or provided a pharmaceutical formulation including an HIVantibody of the invention. When used for in vivo therapy, the antibodiesof the invention are administered to the patient in therapeuticallyeffective amounts (i.e., amounts that eliminate or reduce the patient'sviral burden). The antibodies are administered to a human patient, inaccord with known methods, such as intravenous administration, forexample, as a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. The antibodies can be administered parenterally, whenpossible, at the target cell site, or intravenously. In someembodiments, antibody is administered by intravenous or subcutaneousadministration. Therapeutic compositions of the invention may beadministered to a patient or subject systemically, parenterally, orlocally. The above parameters for assessing successful treatment andimprovement in the disease are readily measurable by routine proceduresfamiliar to a physician.

For parenteral administration, the antibodies may be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable, parenteral vehicle.Examples of such vehicles include, but are not limited, water, saline,Ringer's solution, dextrose solution, and 5% human serum albumin.Nonaqueous vehicles include, but are not limited to, fixed oils andethyl oleate. Liposomes can be used as carriers. The vehicle may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, such as, for example, buffers and preservatives.The antibodies can be formulated in such vehicles at concentrations ofabout 1 mg/ml to 10 mg/ml.

The dose and dosage regimen depends upon a variety of factors readilydetermined by a physician, such as the nature of the infection, forexample, its therapeutic index, the patient, and the patient's history.Generally, a therapeutically effective amount of an antibody isadministered to a patient. In some embodiments, the amount of antibodyadministered is in the range of about 0.1 mg/kg to about 50 mg/kg ofpatient body weight. Depending on the type and severity of theinfection, about 0.1 mg/kg to about 50 mg/kg body weight (for example,about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. The progress ofthis therapy is readily monitored by conventional methods and assays andbased on criteria known to the physician or other persons of skill inthe art. The above parameters for assessing successful treatment andimprovement in the disease are readily measurable by routine proceduresfamiliar to a physician.

Other therapeutic regimens may be combined with the administration ofthe HIV antibody of the present invention. The combined administrationincludes co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities. Suchcombined therapy can result in a synergistic therapeutic effect. Theabove parameters for assessing successful treatment and improvement inthe disease are readily measurable by routine procedures familiar to aphysician.

The terms “treating” or “treatment” or “alleviation” are usedinterchangeably and refer to both therapeutic treatment and prophylacticor preventative measures; wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented. Asubject or mammal is successfully “treated” for an infection if, afterreceiving a therapeutic amount of an antibody according to the methodsof the present invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of infected cells or absence of the infected cells; reductionin the percent of total cells that are infected; and/or relief to someextent, one or more of the symptoms associated with the specificinfection; reduced morbidity and mortality, and improvement in qualityof life issues. The above parameters for assessing successful treatmentand improvement in the disease are readily measurable by routineprocedures familiar to a physician.

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to treat a disease or disorder in a subjector mammal.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include, but not limitedto, buffers such as phosphate, citrate, and other organic acids;antioxidants including, but not limited to, ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such as, butnot limited to, serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as, but not limited to, polyvinylpyrrolidone; amino acidssuch as, but not limited to, glycine, glutamine, asparagine, arginine orlysine; monosaccharides, disaccharides, and other carbohydratesincluding, but not limited to, glucose, mannose, or dextrins; chelatingagents such as, but not limited to, EDTA; sugar alcohols such as, butnot limited to, mannitol or sorbitol; salt-forming counterions such as,but not limited to, sodium; and/or nonionic surfactants such as, but notlimited to, TWEEN; polyethylene glycol (PEG), and PLURONICS.

Where a value of ranges is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

EXAMPLE 1

This example describes materials and methods used in EXAMPLES 2-5 below.

HIV antibodies were cloned and produced following gp140-specific singleB-cell capture as previously described (Mouquet, H. et al. PLoS One 6,e24078 (2011); Tiller, T. et al. J Immunol Methods 329, 112-24 (2008);and Scheid, J. F. et al. Nature 458, 636-40 (2009)). PGT121_(GM) and10-1074_(Gm) “glycomutant” antibodies were generated by substituting10-1074 residues at HC positions 32, 53, 54, 58, 97, 1001 into PGT121and vice versa. Binding properties of anti-gp140 antibodies to HIV Envproteins were assayed by ELISA, SPR and glycan microarray assays aspreviously described (Scheid, J. F. et al. Science 333, 1633-7 (2011);Walker, L. M. et al. Nature 477, 466-70 (2011); and Mouquet, H. et al.PLoS One 6, e24078 (2011)). Neutralization was evaluated using (i) aluciferase-based assay in TZM.b1 cells, and (ii) a PBMC-based assayusing infection with primary HIV-1 variants as previously described (Li,M. et al. J Virol 79, 10108-25 (2005); Euler, Z. et al. Journal ofvirology 85, 7236-45 (2011); and Bunnik, E. M. et al. Nature medicine16, 995-7 (2010)). Structures of PGT121 (“unliganded” and “liganded”),10-1074 and GL Fab fragments were solved by molecular replacement to 2.8Å, 2.3 Å, 1.8 Å and 2.4 Å resolution, respectively.

Single B Cell RT-PCRs and Ig Gene Analyses

Single-cell sorting of gp140⁺CD19⁺IgG⁺ B cells from patient 10 (pt10;referred to as patient 17 in Nature 477(7365):466-470.) PBMCs, cDNAsynthesis and nested PCR amplifications of Ig genes were performed in aprevious study (PLoS One 6(9):e24078). Igλ genes expressed by PGT121clonal variants were PCR amplified using a forward primer (L-Vλ3-21*02:5′ CTGGACCGTTCTCCTCCTCG 3′ (SEQ ID NO: 137)) further upstream in theleader region to avoid the potentially mutated region (31). All PCRproducts were sequenced and analyzed for Ig gene usage, CDR3 analysesand number of VH/V_(κ)somatic hypermutations (IgBLAST and IMGT®).Multiple sequence alignments were performed using the MacVector program(v.12.5.0) with the ClustalW analysis function (default parameters), andwere used to generate dendrograms by the Neighbor Joining method (withBest tree mode and outgroup rooting). Alternatively, dendrograms weregenerated using the UPGMA method (with Best tree mode).

The germline (GL) precursor gene segments of the PGT121-like and10-1074-like antibodies were identified using IgBLAST and IMGT®/V-QUESTas V_(H)4-59*01, J_(H)6*03, V_(L)3-21*02 and J_(L)3*02. (These genesegments are among the most frequently used in the repertoire of humanantibodies (PLoS One 6(8):e22365; Immunogenetics 64(5):337-350). Tobuild a representative GL ancestor sequence, we aligned the IgH and IgLsequences of 10-996 (the antibody containing the fewest somatichypermutations) to the GL sequences using IgBLAST. The GL IgH sequencewas constructed by replacing the mature V_(H) and J_(H) gene segmentswith their GL counterparts and using the 10-996 sequence for the CDRH3region involving N-region nucleotides and the DH gene segment. The GLIgL sequence was assembled from the V_(L)3-21*02 and J_(L)3*02 genesegment sequences.

Cloning and Production of Antibodies

Purified digested PCR products were cloned into human Igγ¹⁻, orIgλ-expressing vectors (J Immunol Methods 329(1-2):112-124). Vectorscontaining IgH and Igλ genes were then sequenced and compared to theoriginal PCR product sequences. PGT121 and 10-303 shared the same Igλgene and had one amino acid difference in position 2 of the IgH gene(FIG. 4); therefore to produce the PGT121 IgG, we used the 10-303 Igλgene and a PGT121 IgH gene generated by introducing a singlesubstitution (V2M) into the 10-303 IgH gene by site-directed mutagenesis(QuikChange Site-Directed Mutagenesis Kit; Stratagene). To generateHis-tagged Fabs, the PGT121 and 10-1074 V_(H) genes were subcloned intoa 6×His-IgCγ1 (‘6×His’ disclosed as SEQ ID NO: 138) expression vectorgenerated by modifying our standard Igyi vector (Science301(5638):1374-1377) to encode the IgG1 CH1 domain followed by a 6×-Histag (SEQ ID NO: 138). IgH DNA fragments encoding PGT121_(GM) (S32Y,K53D, S54R, N58T, H97R, T1001Y) and 10-1074_(GM) (Y32S, D53K, R54S,T58N, R97H, Y1001T) mutant antibodies were obtained as a syntheticminigene (IDT) and subcloned into Igyi-expressing vectors.

Listed below is the heavy chain sequence for 10-1074_(GM) where themutations are underlined. The light chain sequence of 10-1074_(GM) isthe same as that of 10-1074.

(SEQ ID NO: 129) QVQLQESGPGLVKPSETLSVTCSVSGDSMNNSYWTWIRQSPGKGLEWIGYISKSESANYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARHGQRIYGVVSFGEFFTYYSMDVWGKGTTVTVSS

Antibodies and Fab fragments were produced by transient transfection ofIgH and IgL expression plasmids into exponentially growing HEK 293Tcells (ATCC, CRL-11268) using the polyethyleneimine (PEI)-precipitationmethod (PLoS One 6(9):e24078). IgG antibodies were affinity purifiedusing Protein G sepharose beads (GE Healthcare) according to themanufacturer's instructions. Fab fragments were affinity purified usingHisPur™ Cobalt Resin (Thermo scientific) as described below.

HIV-1 Env Proteins

Alanine mutations were introduced into the pYU-2 gp120 vector (gift ofJ. Sodroski, Harvard Medical School) at positions 301 to 303(Asn-Asn-Thr), 324 to 325 (Gly-Asp), and 332 (Asn) (HXBc2 amino acidnumbering) using the QuikChange Site-Directed Mutagenesis kit(Stratagene) according to the manufacturer's instructions. The sameprocedure was used to generate “double glycan” mutants by introducingsingle alanine mutations in the pYU-2 gp120^(N332A) vector at each PNGSlocated between Asn262_(gp120) and Asn406_(gp120). Site-directedmutations were verified by DNA sequencing.

Expression vectors encoding YU-2 gp140 (Journal of virology74(12):5716-5725), YU-2 gp120, HXB2c gp120^(core) (Nature393(6686):648-659), HXB2c 2CCcore (PLoS Pathog 5(5):e1000445) proteins,and YU-2 gp120 mutant proteins were used to transfect HEK 293T cells. Toproduce high-mannose-only YU-2 gp120 protein (gp120_(kif)), 25 μMkifunensine (Enzo Life Sciences) was added at the time of transfection.Culture supernatants were harvested and concentrated usingcentrifugation-based filtration devices (Vivacell 100, Sartorius StedimBiotech Gmbh) that allowed buffer exchange of the samples into 10 mMimidazole, 50 mM sodium phosphate, 300 mM sodium chloride; pH 7.4.Proteins were purified by affinity chromatography using HisPur™ CobaltResin (Thermo scientific) according to the manufacturer's instructions.

For deglycosylation reactions, 50 μg of HEK 293T cell-produced YU-2gp120 in PBS was digested overnight at 37° C. with 200 U of PNGase F(New England Biolabs) or 10,000 U of Endo H_(f) (New England Biolabs) intheir respective reaction buffers without denaturing agents. Afterbuffer exchange into PBS using Centrifugal Filters (Amicon® Ultra,Millipore), glycosidase-treated gp120s (200 ng) were examined bySDS-PAGE using a 4-12% NuPAGE gel (Invitrogen) followed by silverstaining (Pierce Silver Stain Kit, Thermo Scientific).

ELISAs

High-binding 96-well ELISA plates (Costar) were coated overnight with100 ng/well of purified gp120 in PBS. After washing, the plates wereblocked for 2 h with 2% BSA, 1 μM EDTA, 0.05% Tween-PBS (blockingbuffer) and then incubated for 2 h with IgGs at concentrations of 26.7nM (or 427.2 nM for ELISAs using the YU-2 gp120 double glycan mutants)and 7 consecutive 1:4 dilutions in PBS. After washing, the plates weredeveloped by incubation with goat HRP-conjugated anti-human IgGantibodies (Jackson ImmunoReseach) (at 0.8 μg/ml in blocking buffer) for1 h, and by addition of HRP chromogenic substrate (ABTS solution,Invitrogen) (PLoS One 6(9):e24078). Antibody binding to the selectedgp120^(V3) overlapping peptides was tested using a previously describedpeptide-ELISA method.

For competition ELISAs, gp120-coated plates were blocked for 2 h withblocking buffer and then incubated for 2 h with biotinylated antibodies(at a concentration of 26.6 nM for PGT121, 0.21 nM for 10-1074, 0.43 nMfor 10-996 and 1.67 nM for 10-1369) in 1:2 serially diluted solutions ofantibody competitors in PBS (IgG concentration range from 5.2 to 667nM). Plates were developed as described above using HRP-conjugatedstreptavidin (Jackson ImmunoReseach) (at 0.8 μg/ml in blocking buffer).All experiments were performed at least in duplicate.

Glycan Microarray Analysis

Microarrays were generated by robotically printing glycan probes linkedto lipid (neoglycolipids) onto nitrocellulose-coated glass slides(Methods Mol Biol 808:117-136) at two levels (2 and 5 fmol/spot) induplicate. Binding assays were performed with microarrays containing 15neoglycolipids derived from N-glycans of high-mannose and complex-types.The sequences of the probes are shown in FIG. 7A. In brief, antibodieswere tested at 50 μg/ml, and binding was detected with biotinylatedanti-human IgG (Vector) followed by AlexaFluor 647-labeled streptavidin(Molecular Probes).

Surface Plasmon Resonance

Experiments were performed using a Biacore T100 (Biacore, Inc)(Nature467(7315):591-595). Briefly, YU-2 gp140 and gp120 proteins were primaryamine-coupled on CM5 chips (Biacore, Inc.) at a coupling density of 300RUs. Anti-gp120 IgGs and the germline precursor (GL) were injected overflow cells at 1 μM and 10 μM, respectively, at flow rates of 35 μl/minwith 3 min association and 5 min dissociation phases. The sensor surfacewas regenerated by a 30 sec injection of 10 mM glycine-HCl pH 2.5 at aflow rate of 50 μl/min. Dissociation (k_(d) (s⁻¹)), association (k_(a)(M⁻¹ s⁻¹) and binding constants (K_(D) (M) or K_(A) (M⁻¹) werecalculated from kinetic analyses after subtraction of backgrounds usinga 1:1 binding model without a bulk reflective index (RI) correction(Biacore T100 Evaluation software). Binding constants for bivalent IgGscalculated using a 1:1 binding model are referred to in the text as“apparent” affinities to emphasize that the K_(D) values includepotential avidity effects

Neutralization Assays

Virus neutralization was evaluated using a luciferase-based assay inTZM.b1 cells (J Virol 79(16):10108-10125). The HIV-1 pseudovirusestested contained mostly tier-2 and tier-3 viruses (Journal of virology84(3):1439-1452) (Tables 4 and 5). High-mannose-only pseudoviruses wereproduced in wild-type cells treated with 25 μM kifunensine (Enzo LifeSciences) (FIG. 8C) or in HEK 293S GnTI^(−/−) cells (FIG. 8D).Non-linear regression analysis was used to calculate concentrations atwhich half-maximal inhibition was observed (IC₅₀ values). Neutralizationactivities were also evaluated with a previously characterizedPBMC-based assay using infection with primary HIV-1 variants (n=95)isolated from clade B-infected donors with known seroconversion dateseither between 1985 and 1989 (“historical seroconverters”, n=14) orbetween 2003 and 2006 (“contemporary seroconverters”, n=21) (Journal ofvirology 85(14):7236-7245; Nat Med 16(9):995-997). Neutralizationactivity for each antibody was calculated using GraphPad Prism software(v5.0b) as area under the best-fit curve, which fits the proportion ofviruses neutralized over IC₅₀ values ranging from 0.001 to 50 μg/ml.Relative area under the curve (RAUC) values were derived by normalizingall AUC values by the highest value (obtained with 10-1074).

Statistical Analyses

Statistical analyses were performed with the GraphPad Prism software(v5.0b). Neutralization potencies in the TZM-b1 assay against theselected panel of 9 virus strains versus the apparent binding affinitiesof the antibodies for gp120 and gp140 were analyzed using a Spearman'scorrelation test. The Mann Whitney test was used to compare: (i)affinities for gp120/gp140 of antibodies belonging to the PGT121 or10-1074 group, and (ii) neutralization activities against virusesisolated from historical and contemporary seroconverters.

Crystallization and Structure Determinations

6×-His (SEQ ID NO: 138) tagged PGT121, 10-1074 and 10-996GL Fabs forcrystallization were expressed. Fabs were purified from the supernatantsof transiently-transfected HEK 293-6E cells by sequential Ni²⁺-NTAaffinity (Qiagen) and Superdex200 10/300 (GE Healthcare) size exclusionchromatography. For crystals of the unliganded PGT121 Fab, PGT121 IgGwas isolated from the supernatants of transiently-transfected HEK 293-6Ecells by Protein A affinity chromatography (Pierce), and Fab fragmentswere obtained by papain cleavage of the IgG and further purificationusing Superdex200 10/300 (GE Healthcare) size exclusion chromatography.

Purified Fabs were concentrated to 8-20 mg/mL (“unliganded” PGT121, 8mg/mL; 10-1074 and GL, 20 mg/mL) in PBS buffer. The “liganded” PGT121Fab crystals were prepared from a protein sample (final concentration:15 mg/mL) that was mixed with a 3-fold molar excess of NA2 glycan andincubated at 20° C. for 2 hours. Crystallization conditions werescreened at 20° C. using a Mosquito® crystallization robot (TTP labs) in400 nL drops using a 1:1 protein to reservoir ratio. Crystals of“unliganded” PGT121 Fab (P2₁2₁2₁; a=56.8, b=74.7, c=114.9 Å) wereobtained in 24% PEG 4,000, 0.1 M Tris-HCl pH 8.5, 10 mM CuCl₂ andcrystals of “liganded” PGT121 Fab (P2₁2₁2₁; a=67.8, b=67.8, c=94.1 Å)grew in 17% PEG 10,000, 0.1M Bis-Tris pH 5.5, 0.1M CH₃COOHNH₄. Crystalsof 10-1074 Fab (P2₁; a=61.4, b=40.3, c=84.5 Å; β=95.39° were obtained in25% PEG 3,350, 0.1 M Bis-Tris pH 5.5, 0.2 M NaCl, and crystals of GL Fab(P2₁; a=54.9, b=344.7, c=55.2 Å; β91.95° grew in 20% PEG 3,350, 0.24 Msodium malonate pH 7.0, 10 mM MnCl₂. Crystals were cryoprotected bysoaking in mother liquor containing 20% glycerol (“unliganded” and“liganded” PGT121 Fab) or 20% ethylene glycol (10-1074 Fab and GL Fab)and subsequently flash-cooled in liquid nitrogen.

Diffraction data were collected at beamline 12-2 (wavelength=1.029 Å) atthe Stanford Synchrotron Radiation Lightsource (SSRL) on a Pilatus 6Mpixel detector (Dectris). Data were indexed, integrated and scaled usingXDS. Using the data obtained from the “unliganded” PGT121 Fab crystals,we used Phenix to find a molecular replacement solution for one Fab perasymmetric unit (chains H and L for the heavy and light chain,respectively) using two search models, the C_(H)-C_(L) domains of PGT128Fab (PDB code 3PV3) and the V_(H)-V_(L) domains of 2F5 (PDB code 3IDJ)after omitting residues in the CDRH3 and CDRL3 loops. Subsequently, weused the “unliganded” PGT121 structure as a search model to findmolecular replacement solutions for “liganded” PGT121 Fab (one Fab perasymmetric unit), 10-1074 Fab (one Fab per asymmetric unit) and GL (fourFabs per asymmetric unit).

Iterative refinement (including non-crystallographic symmetry restraintsfor GL) was performed using Phenix and manually fitting models intoelectron density maps using Coot. The atomic models were refined to 3.0Å resolution for PGT121 Fab (R_(work)=21.6%; R_(free)=26.4%), 1.9 Åresolution for 10-1074 Fab (R_(work)=18.7%; R_(free)=22.3%), 2.4 Åresolution for four GL Fab molecules (R_(work)=19.4%; R_(free)=23.7%),and 2.4 Å resolution for “liganded” PGT121 Fab (R_(work)=20.1%;R_(free)=24.9%). The atomic model of PGT121 Fab contains 95.2%, 4.9% and0.0% of the residues in the favored, allowed and disallowed regions ofthe Ramachandran plot, respectively (10-1074 Fab: 98.8%, 0.9%, 0.2%; GLFab: 96.0%, 3.8%, 0.23%; “liganded PGT121 Fab: 96.7%, 3.1%, 0.2%). PyMOLwas used for molecular visualization and to generate figures of the Fabstructures. Buried surface area calculations were performed withAreaimol (CCP4 Suite) using a 1.4 Å probe.

Fab structures were aligned using the Super script in PyMOL. Pairwise Cαalignments were performed using PDBeFold.

EXAMPLE 2 Predominance and Diversity of PGT121 Clonotype

gp140-specific IgG memory B cells were isolated from a clade A-infectedAfrican donor using YU-2 gp140 trimers as “bait.” Eighty-seven matchingimmunoglobulin heavy (IgH) and light (IgL) chain genes corresponding to23 unique clonal families were identified. The IgH anti-gp140 repertoirewas dominated by one clonal family representing ˜28% of all expanded Bcell clones. This B cell family corresponds to the same clone asPGT121-123 (Nature 477(7365):466-470) and contained 38 members, 29 ofwhich were unique variants at the nucleotide level (Table 3). Based ontheir IgH nucleotide sequence, the PGT121 family divides into twogroups: a PGT121-like group containing PGT121-123 and 9 closely-relatedvariants, and a second group, 10-1074-like, containing 20 members.Although our traditional primers (J Immunol Methods 329(1-2):112-124;Science 301(5638):1374-1377) did not amplify the IgL genes expressed bythe PGT121 B cell clone due to the nucleotide deletions in the regionencoding framework region 1, 24 of 38 genes were obtained using newIgλ-specific primers designed to amplify heavily somatically-mutatedgenes (Table 3). Consistent with the high levels of hypermutation in theIgH genes (18.2% of the VH gene on average), the amplified Igλ geneswere highly mutated (18.2% of the Vλ gene on average) and carriednucleotide deletions in framework region 1 (FWR1) (12 to 21 nucleotides)and a 9-nucleotide insertion in framework region 3 (FWR3) (FIG. 3B andTable 3).

The sequence alignments of three PGT antibodies (PGT-121, -122, and-123), eleven PGT121 and 10-1074 clonal variants (10-259, 10-303,10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341,10-1369, and 10-1074GM,), likely germline (GL), and consensus sequencesare shown in FIGS. 3(a) and 3(b). The sequences for corresponding heavychain variable regions, light chain variable regions, heavy chain CDRs,and light chain CDRs under both IMGT and KABT systems are listed inTable 1 below. Assigned sequence identification numbers for thesequences under the KABT systems are listed in Table 2 below:

TABLE 1 IgH SEQUENCES IMGT FWR1 CDR1 FWR2 CDR2 FWR3 CDR3 FWR4 10-1369QVQLQESGPGLVKPLETLSLTCN GAFIADHY WSWIRLPLGKG VHDSGDINYNPSLKNRVHLSLDKSTNQVSLKLM ATTKHGRRIYGVVAFGE WGRGTTVTVSS (SEQ ID VS(SEQ ID NO: PEWIGY (SEQ ID AVTAGDSALYYC WFTYFYMDV (SEQ ID NO: 23) 139)NO: 140) NO: 141) 10-259 QVHLQESGPGLVKPSETLSLTCN GTLVRDNY WSWMRQPLGKQVHDSGDT NYNPSLKSRVHLSLDKSNNLVSLRLT ATTKHGRRIYGIVAFNE WGKGTTVTVSS (SEQ IDVS (SEQ ID NO: PEWIGY (SEQ ID AVTAADSATYYC WFTYFYMDV (SEQ ID NO: 3) 142)NO: 143) NO: 144) 10-303 QVQLQESGPGLVKPSETLSLTCS GASISDSY WSWIRRSPGKGVHKSGDT NYSPSLKSRVNLSLDTSKNQVSLSLV ARTLHGRRIYGIVAFNE WGNGTQVTVSS (SEQ IDVS (SEQ ID NO: LEWIGY (SEQ ID AATAADSGKYYC WFTYFYMDV (SEQ ID NO: 5) 145)NO: 146) NO: 147) 10-410 QVQLQESGPGLVKPPETLSLTCS GASVNDAY WSWIRQSPGKRVHHSGDT NYNPSLKRRVTFSLDTAKNEVSLKLV ARALHGKRIYGIVALGE WGKGTTVTVSS (SEQ IDVS (SEQ ID NO: PEWVGY (SEQ ID ALTAADSAVYFC LFTYFYMDV (SEQ ID NO: 7) 148)NO: 149) NO: 150) 10-1130 QVQLQESGPGLVKPPETLSLTCS GASINDAY WSWIRQSPGKRVHHSGDT NYNPSLKRRVTFSLDTAKNEVSLKLV ARALHGKRIYGIVALGE WGKGTTVTVSS (SEQ IDVS (SEQ ID NO: PEWVGY (SEQ ID DLTAADSAVYFC LFTYFYMDV (SEQ ID NO: 17)151) NO: 152) NO: 153) 10-1121 QVQLQESGPGLVKPPETLSLTCS GASINDAYWSWIRQSPGKR VHHSGDT NYNPSLKRRVSFSLDTAKNEVSLKLV ARALHGKRIYGIVALGEWGKGTTVTVSS (SEQ ID VS (SEQ ID NO: PEWVGY (SEQ ID DLTAADSAIYFCLFTYFYMDV (SEQ ID NO: 15) 154) NO: 155) NO: 156) 10-1146QVQLVESGPGLVTPSETLSLTCT NGSVSGRF WSWIRQSPGRG FSDTDRSEYSPSLRSRLTLSLDASRNQLSLKLK ARAQQGKRIYGIVSFGE WGKGTAVTVSS (SEQ ID VS(SEQ ID NO: LEWIGY (SEQ ID SVTAADSATYYC FFYYYYMDA (SEQ ID NO: 19) 157)NO: 158) NO: 159) 10-996 QVQLQESGPGLVKPSETLSLTCS NGSVSGRF WSWIRQSPGRGFSDTEKS NYNPSLRSRLTLSVDASKNQLSLKLN ARTQQGKRIYGVVSFGE WGKGTAVTVSS (SEQ IDVS (SEQ ID NO: LEWIGY (SEQ ID SVTAADSATYYC FFHYYYMDA (SEQ ID NO: 11)160) NO: 161) NO: 162) GL (SEQ QVQLQESGPGLVKPSETLSLTCT GGSISSYYWSWIRQPPGKG IYYSGST NYNPSLKSRVTISVDTSKNQFSLKLS ARTQQGKRIYGVVSFGDWGKGTTVTVSS ID NO: VS (SEQ ID NO: LEWIGY (SEQ ID SVTPADTAVYYCYYYYYYMDV (SEQ ID 31) 163) NO: 164) NO: 165) 10-1341QVQLQESGPGLVKPSETLSVTCS GDSMNNYY WTWIRQSPGKG ISDRESATYNPSLNSRVVISRDTSTNQLSLKLN ATARRGQRIYGVVSFGE WGRGTTVTVSS (SEQ ID VS(SEQ ID NO: LEWIGY (SEQ ID SVTPADTAVYYC FFYYYSMDV (SEQ ID NO: 21) 166)NO: 167) NO: 168) 10-847 QVQLQESGPGLVKPSETLSVTCS GDSMNNYY WTWIRQSPGKGISDRASA TYNPSLNSRVVISRDTSKNQLSLKLN ATARRGQRIYGVVSFGE WGKGTTVTVSS (SEQ IDVS (SEQ ID NO: LEWIGY (SEQ ID SVTPADTAVYYC FFYYYSMDV (SEQ ID NO: 9) 169)NO: 170) NO: 171) 10-1074 QVQLQESGPGLVKPSETLSVTCS GDSMNNYY WTWIRQSPGKGISDRESA TYNPSLNSRVVISRDTSKNQLSLKLN ATARRGQRIYGVVSFGE WGKGTTVTVSS (SEQ IDVS (SEQ ID NO: LEWIGY (SEQ ID SVTPADTAVYYC FFYYYSMDV (SEQ ID NO: 13)172) NO: 173) NO: 174) 10- QVQLQESGPGLVKPSETLSVTCS GDSMNNSY WTWIRQSPGKGISKSESA NYNPSLNSRVVISRDTSKNQLSLKLN ATARHGQRIYGVVSFGE WGKGTTVTVSS 1074GMVS (SEQ ID NO: LEWIGY (SEQ ID SVTPADTAVYYC FFTYYSMDV (SEQ ID (SEQ ID175) NO: 176) NO: 177) NO: 129) RABAT FWR1 CDR1 FWR2 CDR2 FWR3 CDR3 FWR410-1369 QVQLQESGPGLVKPLETLSLTCN DHYWS (SEQ WIRLPLGKGPE YVHDSGDINYRVHLSLDKSTNQVSLKLMAVTAGDSA TKHGRRIYGVVAFGEWF WGRGTTVTVSS (SEQ ID VSGAFIAID NO: 99) WIG NPSLKN LYYCAT TYFYMDV (SEQ ID NO: 23) (SEQ ID NO: 101)NO: 100) 10-259 QVHLQESGPGLVKPSETLSLTCN DNYWS (SEQ WMRQPLGKQPEYVHDSGDTNY RVHLSLDKSNNLVSLRLTAVTAADSA TKHGRRIYGIVAFNEWF WGKGTTVTVSS(SEQ ID VSGTLVR ID NO: 39) WIG NPSLKS TYYCAT TYFYMDV (SEQ ID NO: 3)(SEQ ID NO: 41) NO: 40) 10-303 QVQLQESGPGLVKPSETLSLTCS DSYWS (SEQWIRRSPGKGLE YVHKSGDTNY RVNLSLDTSKNQVSLSLVAATAADSG TLHGRRIYGIVAFNEWFWGNGTQVTVSS (SEQ ID VSGASIS ID NO: 45) WIG SPSLKS KYYCAR TYFYMDV (SEQ IDNO: 5) (SEQ ID NO: 47) NO: 46) 10-410 QVQLQESGPGLVKPPETLSLTCS DAYWS (SEQWIRQSPGKRPE YVHHSGDTNY RVTFSLDTAKNEVSLKLVALTAADSA ALHGKRIYGIVALGELFWGKGTTVTVSS (SEQ ID VSGASVN ID NO: 51) WVG NPSLKR VYFCAR TYFYMDV (SEQ IDNO: 7) (SEQ ID NO: 53) NO: 52) 10-1130 QVQLQESGPGLVKPPETLSLTCSDAYWS (SEQ WIRQSPGKRPE YVHHSGDTNY RVTFSLDTAKNEVSLKLVDLTAADSAALHGKRIYGIVALGELF WGKGTTVTVSS (SEQ ID VSGASIN ID NO: 81) WVG NPSLKRVYFCAR TYFYMDV (SEQ ID NO: 17) (SEQ ID NO: 83) NO: 82) 10-1121QVQLQESGPGLVKPPETLSLTCS DAYWS (SEQ WIRQSPGKRPE YVHHSGDTNYRVSFSLDTAKNEVSLKLVDLTAADSA ALHGKRIYGIVALGELF WGKGTTVTVSS (SEQ ID VSGASINID NO: 75) WVG NPSLKR IYFCAR TYFYMDV (SEQ ID NO: 15) (SEQ ID NO: 77)NO: 76) 10-1146 QVQLVESGPGLVTPSETLSLTCT GRFWS (SEQ WIRQSPGRGLEYFSDTDRSEY RLTLSLDASRNQLSLKLKSVTAADSA AQQGKRIYGIVSFGEFF WGKGTAVTVSS(SEQ ID VSNGSVS ID NO: 87) WIG SPSLRS TYYCAR YYYYMDA (SEQ ID NO: 19)(SEQ ID NO: 89) NO: 88) 10-996 QVQLQESGPGLVKPSETLSLTCS GRFWS (SEQWIRQSPGRGLE YFSDTEKSNY RLTLSVDASKNQLSLKLNSVTAADSA TQQGKRIYGVVSFGEFFWGKGTAVTVSS (SEQ ID VSNGSVS ID NO: 63) WIG NPSLRS TYYCAR HYYYMDA (SEQ IDNO: 11) (SEQ ID NO: 65) NO: 64) GL (SEQ QVQLQESGPGLVKPSETLSLTCTSYYWS (SEQ WIRQPPGKGLE YIYYSGSTNY RVTISVDTSKNQFSLKLSSVTAADTATQQGKRIYGVVSFGDYY WGKGTTVTVSS ID NO: VSGGSIS ID NO: 123) WIG NPSLKSVYYCAR YYYYMDV (SEQ ID 31) (SEQ ID NO: 125) NO: 124) 10-1341QVQLQESGPGLVKPSETLSVTCS NYYWT (SEQ WIRQSPGKGLE YISDRESATYRVVISRDTSTNQLSLKLNSVTPADTA ARRGQRIYGVVSFGEFF WGRGTTVTVSS (SEQ ID VSGDSMNID NO: 93) WIG NPSLNS VYYCAT YYYSMDV (SEQ ID NO: 21) (SEQ ID NO: 95)NO: 94) 10-847 QVQLQESGPGLVKPSETLSVTCS NYYWT (SEQ WIRQSPGKGLE YISDRASATYRVVISRDTSKNQLSLKLNSVTPADTA ARRGQRIYGVVSFGEFF WGKGTTVTVSS (SEQ ID VSGDSMNID NO:57)  WIG NPSLNS VYYCAT YYYSMDV (SEQ ID NO: 9) (SEQ ID NO: 59)NO: 58) 10-1074 QVQLQESGPGLVKPSETLSVTCS NYYWT (SEQ WIRQSPGKGLEYISDRESATY RVVISRDTSKNQLSLKLNSVTPADTA ARRGQRIYGVVSFGEFF WGKGTTVTVSS(SEQ ID VSGDSMN ID NO: 69) WIG NPSLNS VYYCAT YYYSMDV (SEQ ID NO: 13)(SEQ ID NO: 71) NO: 70) 10- QVQLQESGPGLVKPSETLSVTCS NSYWT (SEQWIRQSPGKGLE YISKSESANY RVVISRDTSKNQLSLKLNSVTPADTA ARHGQRIYGVVSFGEFFWGKGTTVTVSS 1074GM VSGDSMN ID NO: 131) WIG NPSLNS VYYCAT TYYSMDV (SEQ ID(SEQ ID (SEQ ID NO: 133) NO: NO: 132) 129) IgL SEQUENCES IMGT FWR1 CDR1FWR2 CDR2 FWR3 CDR3 FWR4 GL (SEQ SYVLTQPPSVSVAPGQTARITCG NIGSKS (SEQVHWYQQKPGQA DDS (SEQ DRPSGIPERFSGSNSGNTATLTISRV QVWDSSSDHPWV (SEQFGGGTKLTVL ID NO: GN ID NO: 178) PVLVVY ID NO: EAGDEADYYC ID NO: 180)32) 179) 10-1369 SSMSVSPGETAKITCGEK SIGSRA (SEQ VQWYQKKPGQP NNQ (SEQDRPSGVPERFSASPDIEFGTTATLTI HIYDARRPTNWV (SEQ FDRGTILTVL (SEQ IDID NO: 181) PSLIIY ID NO: TNVEAGDEADYYC ID NO: 183) NO: 24) 182) 10-259SSMSVSPGETAKISCGKE SIGSRA (SEQ VQWYQQKSGQP NNQ (SEQDRPSGVPERFSATPDFGAGITAILTI HIYDARGGTNWV (SEQ FDRGATLIVL (SEQ IDID NO: 184) PSLIIY ID NO: TNVEADDEADYYC ID NO: 186) NO: 4) 185) 10-303SDISVAPGETARISCGEK SLGSRA (SEQ VQWYQHRAGQA NNQ (SEQDRPSGIPERFSGSPDSPFGTTATLTI HIWDSRVPIKWV (SEQ FGGGTTLTVL (SEQ IDID NO: 187) PSLIIY ID NO: TSVEAGDEADYYC ID NO: 189) NO: 6) 188) 10-1121SFVSVAPGQTARITCGEE SLGSRS (SEQ VIWYQQRPGQA NNH (SEQDRPSGIPERFSGSPGSTFGTTATLTI HIWDSRRPINWV (SEQ FGEGTTLTVL (SEQ IDID NO: 190) PSLIMY ID NO: TSVEAGDEADYYC ID NO: 192) NO: 16) 191) 10-410SFVSVAPGQTARITCGEE SLGSRS (SEQ VIWYQQRPGQA NNN (SEQDRPSGIPERFSGSPGSTFGTTATLTI HIWDSRRPINWV (SEQ FGEGTTLTVL (SEQ IDID NO: 193) PSLIIY ID NO: TSVEAGDEADYYC ID NO: 195) NO: 8) 194) 10-1130SFVSVAPGQTARITCGEE SLGSRS (SEQ VIWYQQRPGQA NNN (SEQDRPSGIPERFSGSPGSTFGTTATLTI HIWDSRRPINWV (SEQ FGEGTTLTVL (SEQ IDID NO: 196) PSLIIY ID NO: TSVEAGDEADYYC ID NO: 198) NO: 18) 197) 10-847SYVRPLSVALGETASISCGRQ ALGSRA (SEQ VQWYQHRPGQA NNQ (SEQDRPSGIPERFSGTPDINFGTRATLTI HMWDSRSGFSWS (SEQ FGGATRLTVL (SEQ IDID NO: 199) PILLIY ID NO: SGVEAGDEADYYC ID NO: 201) NO: 10) 200) 10-1074SYVRPLSVALGETARISCGRQ ALGSRA (SEQ VQWYQHRPGQA NNQ (SEQDRPSGIPERFSGTPDINFGTRATLTI HMWDSRSGFSWS (SEQ FGGATRLTVL (SEQ IDID NO: 202) PILLIY ID NO: SGVEAGDEADYYC ID NO: 204) NO: 14) 203) 10-1341SYVRPLSVALGETARISCGRQ ALGSRA (SEQ VQWYQHRPGQA NNQ (SEQDRPSGIPERFSGTPDINFGTRATLTI HMWDSRSGFSWS (SEQ FGGATRLTVL (SEQ IDID NO: 205) PILLIY ID NO: SGVEAGDEADYYC ID NO: 207) NO: 22) 206) 10-996SSLPLSVAPGATAKIACGEK SFASRA (SEQ VQWYQQKPGQA NNQ (SEQDRPAGVSERFSGTPDVGFGSTATLTI HKWDSRSPLSWV (SEQ FGGGTQLTVL (SEQ IDID NO: 208) PVLIIY ID NO: SRVEAGDEADYYC ID NO: 210) NO: 12) 209) 10-1146SSLPLSLAPGATAKIPCGEK SRGSRA (SEQ VQWYQQKPGQA NNQ (SEQDRPAGVSERYSGNPDVAIGVTATLTI HYWDSRSPISWV (SEQ FGGWTQLTVL (SEQ IDID NO: 211) PTLIIY ID NO: SRVEAGDEAEYYC ID NO: 213) NO: 20) 212) RABATFWR1 CDR1 FWR2 CDR2 FWR3 CDR3 FWR4 GL (SEQ SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVH WYQQKPGQAPV DDSDRPS GIPERFSGSNSGNTATLTISRVEAGDQVWDSSSDHPWV (SEQ FGGGTKLTVL ID NO: (SEQ ID NO: LVVY (SEQ ID EADYYCID NO: 128) 32) 126) NO: 127) 10-1369 SSMSVSPGETAKITC GEKSIGSRAVQWYQKKPGQPPS NNQDRPS GVPERFSASPDIEFGTTATLTITNVE HIYDARRPTNWV (SEQFDRGTILTVL (SEQ ID (SEQ ID NO: LIIY (SEQ ID AGDEADYYC ID NO: 104)NO: 24) 102) NO: 103) 10-259 SSMSVSPGETAKISC GKESIGSRAVQ WYQQKSGQPPSNNQDRPS GVPERFSATPDFGAGITAILTITNVE HIYDARGGTNWV (SEQ FDRGATLIVL (SEQ ID(SEQ ID NO: LIIY (SEQ ID ADDEADYYC ID NO: 44) NO: 4) 42) NO: 43) 10-303SDISVAPGETARISC GEKSLGSRAVQ WYQHRAGQAPS NNQDRPSGIPERFSGSPDSPFGTTATLTITSVE HIWDSRVPIKWV (SEQ FGGGTTLTVL (SEQ ID(SEQ ID NO: LIIY (SEQ ID AGDEADYYC ID NO: 50) NO: 6) 48) NO: 49) 10-1121SFVSVAPGQTARITC GEESLGSRSVI WYQQRPGQAPS NNHDRPSGIPERFSGSPGSTFGTTATLTITSVE HIWDSRRPINWV (SEQ FGEGTTLTVL (SEQ ID(SEQ ID NO: LIMY (SEQ ID AGDEADYYC ID NO: 80) NO: 16) 78) NO: 79) 10-410SFVSVAPGQTARITC GEESLGSRSVI WYQQRPGQAPS NNNDRPSGIPERFSGSPGSTFGTTATLTITSVE HIWDSRRPINWV (SEQ FGEGTTLTVL (SEQ ID(SEQ ID NO: LIIY (SEQ ID AGDEADYYC ID NO: 56) NO: 8) 54) NO: 55) 10-1130SFVSVAPGQTARITC GEESLGSRSVI WYQQRPGQAPS NNNDRPSGIPERFSGSPGSTFGTTATLTITSVE HIWDSRRPINWV (SEQ FGEGTTLTVL (SEQ ID(SEQ ID NO: LIIY (SEQ ID AGDEADYYC ID NO: 86) NO: 18) 84) NO: 85) 10-847SYVRPLSVALGETASISC GRQALGSRAVQ WYQHRPGQAPI NNQDRPSGIPERFSGTPDINFGTRATLTISGVE HMWDSRSGFSWS (SEQ FGGATRLTVL (SEQ ID(SEQ ID NO: LLIY (SEQ ID AGDEADYYC ID NO: 62) NO: 10) 60) NO: 61)10-1074 SYVRPLSVALGETARISC GRQALGSRAVQ WYQHRPGQAPI NNQDRPSGIPERFSGTPDINFGTRATLTISGVE HMWDSRSGFSWS (SEQ FGGATRLTVL (SEQ ID(SEQ ID NO: LLIY (SEQ ID AGDEADYYC ID NO: 74) NO: 14) 72) NO: 73)10-1341 SYVRPLSVALGETARISC GRQALGSRAVQ WYQHRPGQAPI NNQDRPSGIPERFSGTPDINFGTRATLTISGVE HMWDSRSGFSWS (SEQ FGGATRLTVL (SEQ ID(SEQ ID NO: LLIY (SEQ ID AGDEADYYC ID NO: 98) NO: 22) 96) NO: 97) 10-996SSLPLSVAPGATAKIAC GEKSFASRAVQ WYQQKPGQAPV NNQDRPAGVSERFSGTPDVGFGSTATLTISRVE HKWDSRSPLSWV (SEQ FGGGTQLTVL (SEQ ID(SEQ ID NO: LIIY (SEQ ID AGDEADYYC ID NO: 68) NO: 12) 66) NO: 67)10-1146 SSLPLSLAPGATAKIPC GEKSRGSRAVQ WYQQKPGQAPT NNQDRPAGVSERYSGNPDVAIGVTATLTISRVE HYWDSRSPISWV (SEQ FGGWTQLTVL (SEQ ID(SEQ ID NO: LIIY (SEQ ID AGDEAEYYC ID NO: 92) NO: 20) 90) NO: 91)

TABLE 2 SEQ ID NOs Variable Region CDRs 1-3 Name Heavy chain (H) Lightchain (L) H L consensus SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NOs: 33-35 SEQID NOs: 36-38 10-259 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NOs: 39-41 SEQ IDNOs: 42-44 10-303 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NOs: 45-47 SEQ IDNOs: 48-50 10-410 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NOs: 51-53 SEQ IDNOs: 54-56 10-847 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NOs: 57-59 SEQ IDNOs: 60-62 10-996 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NOs: 63-65 SEQ IDNOs: 66-68 10-1074 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NOs: 69-71 SEQ IDNOs: 72-74 10-1121 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NOs: 75-77 SEQ IDNOs: 78-80 10-1130 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NOs: 81-83 SEQ IDNOs: 84-86 10-1146 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NOs: 87-89 SEQ IDNOs: 90-92 10-1341 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NOs: 93-95 SEQ IDNOs: 96-98 10-1369 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NOs: 99-101 SEQ IDNOs: 102-104 PGT-121 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NOs: 105-107 SEQID NOs: 108-110 PGT-122 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NOs: 111-113SEQ ID NOs: 114-116 PGT-123 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NOs:117-119 SEQ ID NOs: 120-122 GL SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NOs:123-125 SEQ ID NOs: 126-128 10-1074GM SEQ ID NO: 129 SEQ ID NO: 130 SEQID NOs: 131-133 SEQ ID NOs: 134-136

Eleven new unique variants were expressed (Table 3) and demonstratedbinding to YU-2 gp120 and gp140 by ELISA and surface plasmon resonance(SPR). Unless otherwise noted, the gp120 and gp140 proteins for theseand other experiments were expressed in mammalian cells that can attacheither a complex-type or a high-mannose N-glycan to a PNGS. The level ofreactivity with gp120 differed between antibodies belonging to thePGT121 and 10-1074 groups, the latter exhibiting higher apparentaffinities (FIG. 3A) mainly due to slower dissociation from gp120/gp140for the 10-1074-related antibodies (FIG. 4B).

EXAMPLE 3 PGT121 and 10-1074 Epitopes

Asn332_(gp120) in the vicinity of the V3 loop stem was reported ascritical for binding and viral neutralization by PGT121 (Nature477(7365):466-470), thus we examined the role of V3 in antigenrecognition by PGT121-like and 10-1074-like antibodies. ELISAs wereperformed using HXB2 gp120 “core” proteins that lack V1-V3 loops(gp120^(core)) or retain a portion of V3 (2CC-core), and using a YU-2gp120 mutant protein carrying a double alanine substitution in the V3stem (gp120^(GD324-5AA)). The tested antibodies showed decreasedreactivity against variants lacking the V3 loop and gp120^(GD324-5AA)when compared to intact YU-2 gp120, with the binding of 10- 1074-groupantibodies being the most affected (FIGS. 5A and B). These resultssuggest that recognition by both antibody groups involves proteindeterminants in the vicinity of the V3 loop. None of the antibodiesbound to overlapping peptides spanning V3, suggesting the targetedepitopes are discontinuous and/or require a particular conformation notachieved by isolated peptides (FIG. 5C).

Asn332_(gp120) (Asn337_(gp120) in earlier numbering (J Proteome Res7(4):1660-1674)) is the N-terminal residue of a potentialN-glycosylation site (PNGS) defined as the sequence Asn-X-Ser/Thr. Todetermine whether Asn332_(gp120) and/or its N-linked glycan are requiredfor gp120 reactivity of the new PGT121- and 10-1074-group antibodies, wetested their binding to YU-2 gp120^(N332A) by ELISA. The N332Asubstitution diminished the binding of PGT121 and all the new antibodyvariants, whereas their reactivity against a mutant gp120 lacking anearby glycosylation site (gp120^(NNT301-3AAA) mutant) was unchanged. Todetermine if a PNGS in addition to the Asn332_(gp120) PNGS affectsrecognition by the new antibodies, we constructed a series of 11 doubleglycan mutants in which the N332A mutation in YU-2 gp120 was combinedwith mutation of PNGSs located between Asn262_(gp120) andAsn406_(gp120). All of the PGT121-like and 10-1074-like antibodies boundto each of the double glycan mutants with comparable affinity as to thatfor gp120^(N332A).

To compare overall glycan recognition by the PGT121- and 10-1074-likeantibodies, we examined their binding to YU-2 gp120 treated with PNGaseF, which cleaves both complex-type and high-mannose N-glycans. Becausegp120 cannot be fully deglycosylated enzymatically unless it isdenaturated, PNGase F treatment resulted in partial deglycosylation ofnatively-folded gp120 (FIG. 6). Nevertheless, the reactivities of thetwo groups of antibodies differed in that partial deglycosylation ofgp120 by PNGase F decreased the binding activity of all PGT121-likeantibodies but none of the 10-1074-like antibodies (FIG. 6C). Similarexperiments conducted with YU-2 gp120 treated with Endo H, which cleaveshigh-mannose, but not complex-type, N-glycans, affected binding of10-1074-like antibodies more than PGT121-like antibodies (FIG. 6D).

An N-glycan microarray revealed that six of seven tested PGT121-likeantibodies showed detectable binding to complex-type mono- orbi-antennary N-glycans terminating with galactose or α2-6-linked sialicacid but no detectable binding to high-mannose type glycans,corroborating and extending previous reports of no binding of PGT121-123to high-mannose N-glycans and no competition by Man₄ and Mang dendronsfor gp120 binding (FIG. 7). In contrast, there was no detectable bindingto protein-free glycans by 10-1074-like antibodies (FIG. 7). AlthoughPGT121-like antibodies bound to protein-free complex-type, but nothigh-mannose, N-glycans, PGT121-like antibodies retained binding to YU-2gp120 produced in cells treated with kifunensine (gp120_(kif)), amannosidase inhibitor that results in exclusive attachment ofhigh-mannose glycans to PNGSs (FIG. 8B). Most of the PGT121-likeantibodies exhibited a small, but reproducible, decrease in binding togp120_(kif). By contrast, 10-1074-like antibodies retained full bindingto gp120_(kif) (FIG. 8B). These results are consistent with thehypothesis that high-mannose, as well as complex-type, N-glycans can beinvolved in the epitope of PGT121-like antibodies.

Epitope mapping experiments were performed with two representativemembers of each group (PGT121 and 10-1369 for the PGT121-like group;10-1074 and 10-996 for the 10-1074-like group) by competition ELISA. Allfour antibodies showed cross-competition, but PGT121 more modestlyinhibited the binding of 10-996 and 10-1074 to gp120 than vice-versa. Tofurther map the targeted epitopes, we used anti-gp120 antibodies thatrecognize the crown of the V3 loop (FIG. 5), the CD4bs, the co-receptorbinding site (CD4-induced; CD4_(i)), a constellation of high-mannoseN-glycans (2G12) (Journal of virology 76(14):7293-7305; Proc Natl AcadSci USA 102(38):13372-13377)), or the V3 loop and N-linked glycans atpositions 301 and 332 (PGT128). Anti-V3 crown antibodies inhibited thebinding of PGT121 and 10-1369 but did not interfere with the binding of10-996 and 10-1074. PGT128, and to a lesser extent 2G12, but not theCD4bs and CD4i antibodies, diminished the binding of all four antibodiesto gp120.

Taken together, these data suggest that PGT121 clonal members recognizea site involving a protein determinant in the vicinity of the V3 loopand the Asn332_(gp120)-associated glycan. However, the clone segregatesinto two families, the PGT121-like and 10-1074-like groups, which differin their affinities for gp120 and in the role of glycans in epitopeformation.

EXAMPLE 4 Broad and Potent HIV Neutralization

To evaluate the neutralizing activity of the new PGT121 variants, wemeasured their ability to inhibit HIV infection of TZM-b1 cells using 10viral strains including R1166.c1, which lacks the PNGS at gp120 position332. All PGT121 variants, including the 10-1074-like antibodies,neutralized 9 pseudoviruses and none neutralized the R1166.c1 control(FIG. 1A and Table 4). Neutralizing activity correlated with affinityfor the HIV spike, with the 10-1074 group showing slightly greaterpotencies than the PGT121 group (FIG. 1B and FIG. 4C). A representativegermline version (GL) of the PGT121/10-1074 antibody clonotype failed tobind gp120/gp140 or neutralize any viruses in the panel, implying thatsomatic mutation is required for binding and neutralization. Pairing GLlight chains with mutated 10-1074- or 10-996-group heavy chains failedto rescue binding or neutralization, suggesting that both mutated chainscontribute to proper assembly of the antibody paratope.

Next assays were carried out to compared the neutralization activitiesof PGT121 and two 10-1074-like antibodies (10-996 and 10-1074) againstan extended panel of 119 difficult-to-neutralize pseudoviruses(classified as tier-2 and tier-3) (Tables 4 and 5). 10-996 and 10-1074showed neutralization potencies and breadth similar to PGT121 (FIG. 1C,FIG. 9, and Tables 5 and 6). As anticipated, most viruses bearing aminoacid changes at gp120 positions 332 and/or 334 (spanning theAsn332-X-Ser334/Thr334 PNGS) were resistant to neutralization (83.8%were resistant to PGT121, 100% were resistant to 10-1074 and 10-996).Mutation at this PNGS accounted for the majority of viruses resistant toneutralization (68.5% for 10-996, 72.5% for 10-1074 and 60.8% forPGT121) (Table 7). Comparable neutralization activities were observedfor the IgG and Fab forms of PGT121 and 10-1074, suggesting thatbivalency is not critical for their activity (FIG. 1D).

To evaluate the potential role of complex-type N-glycans on the HIVenvelope in neutralization by PGT121 and 10-1074, we producedhigh-mannose-only virions in two different ways: by assemblingpseudoviruses in cells treated with kifunensine, which results inMan₉GlcNAc₂ N-linked glycans, or by assembly in HEK 293S GnTI^(−/−)cells, which results in Man₅GlcNAc₂ N-linked glycans. We found thatPGT121 neutralized 2 of 3 kifunensine-derivedPGT121-sensitive/10-1074-resistant strains equivalently to theircounterparts produced in wildtype cells (FIG. 8C). TwoPGT121-sensitive/10-1074-sensitive viral strains produced in GnTI^(−/−)cells were equally as sensitive to PGT121 and 10-1074 as theircounterparts produced in wildtype cells. Consistent with previousreports that complex-type N-glycans partially protect the CD4 bindingsite from antibody binding, the viruses produced in GnTI^(−/−) cellswere more sensitive to CD4-binding site antibodies (NIH45-46^(G54W) and3BNC60) (FIG. 8D).

EXAMPLE 5 Newly-Transmitted HIV-1

We next examined the activity of PGT121 and 10-1074 against transmittedfounder viruses by evaluating neutralization in a peripheral bloodmononuclear cell (PBMC)-based assay using 95 clade B viruses isolatedfrom a cohort of individuals who seroconverted between 1985 and 1989(historical seroconverters, n=14) or between 2003 and 2006 (contemporaryseroconverters, n=25) (51, 52). We compared PGT121 and 10-1074 withanti-CD4bs bNAbs and other bNAbs including VRC01, PG9/PG16, b12, 2G12,4E10 and 2F5. Clustering analyses of neutralization activity showedsegregation into two groups; the PGT121/10-1074 group contained the mostactive HIV neutralizers including the anti-CD4bs and PG9 antibodies(Table 8). Remarkably, 10-1074 showed exceptional neutralization potencyon this clade B virus panel, exhibiting the greatest breadth at 0.1μg/ml (67% of the 95 clade B viruses) of all bNAbs tested (Table 8).Although 10-1074 showed higher potency on contemporary clade B virusesthan PGT121 (˜20-fold difference), both antibodies were more effectiveagainst historical than contemporary viruses (FIG. 1E and FIG. 10).

EXAMPLE 6 Crystal Structures of PGT121, 10-1074 and GL

To investigate the structural determinants of the differences betweenPGT121-like and 1074-like antibodies, we solved crystal structures ofthe Fab fragments of PGT121, 10-1074 and a representative germlineprecursor (GL) at 3.0 Å, 1.9 Å and 2.4 Å resolution, respectively (Table9). Superimposition of the heavy and light chain variable domains (V_(H)and V_(L)) among the three Fabs showed conservation of the backbonestructure, with differences limited to small displacements of the CDRH3and CDRL3 loops of the affinity-matured Fabs relative to GL (Table 10).

An unusual feature shared by the antibodies is their long (25 residues)CDRH3 loop, which forms a two-stranded anti-parallel β-sheet extendingthe V_(H) domain F and G strands. In each Fab, the tip of the extendedCDRH3 loop primarily contains non-polar residues. A similar structuralfeature was observed for the CDRH3 of PGT145, a carbohydrate-sensitiveantibody whose epitope involves the gp120 V1V2 loop. However, theextended two-stranded β-sheet of PGT145's CDRH3 contains mostlynegatively-charged residues, including two sulfated tyrosines at thetip. Aligning V_(H)-V_(L) of PGT121 and PGT145 (Table 10) shows thatCDRH3_(PGT145) extends past CDRH3_(PGT121) and that its tip and V_(H)domain are aligned, whereas the CDRH3s of PGT121, 10-1074 and GL tilttowards V_(L). The tilting of CDRH3_(PGT121)/CDRH3₁₀₋₁₀₇₄/CDRH3_(GL)towards V_(L) opens a cleft between CDRH2 and CDRH3, a feature notshared by related antibodies.

PGT121 and 10-1074 are highly divergent with respect to GL and eachother (of 132 residues, PGT121_(VH) differs from 10-1074_(VH) andGL_(VH) by 36 and 45 residues, respectively, and 10-1074_(VH) andGL_(VH) differ by 29). The majority of the PGT121/10-1074 differencesare located in the CDR_(VH) loops and CDRL3. Interestingly, sixsubstitutions in CDRH3 (residues 100d, 100f, 100h, 100j, 100l, 100n)alternate such that every second residue is substituted, causingresurfacing of the cleft between CDRH2 and CDRH3 that results from CDRH3tilting towards V_(L). This region likely contributes to the differentfine specificities of PGT121 and 10-1074. Five other solvent-exposedsubstitutions in heavy chain framework region 3 (FWR3_(HC)) (residues64, 78, 80-82; strands D and E) are potential antigen contact sitesgiven that framework regions in HIV antibodies can contact gp120. Otherdifferences that may contribute to fine specificity differences includea negative patch on PGT121 in the vicinity of Asp56_(HC) not present in10-1074 or GL (Ser56_(HC) in 10-1074 and GL) and positive patches on theCDRL1 and CDRL3 surface not found on the analogous surface of GL.

Somatic mutations common to PGT121 and 10-1074 may be involved in sharedfeatures of their epitopes. The heavy chains of PGT121 and 10-1074 shareonly three common mutations (of 36 PGT121-GL and 29 10-1074-GLdifferences). In contrast, PGT121 and 10-1074 share 18 common lightchain mutations (of 37 PGT121-GL and 36 10-1074-GL differences),including an insertion in light chain FWR3 that causes bulging of theloop connecting strands D and E, and the substitution ofAsp50_(LC)-Asp51_(LC) in CDRL2_(GL) to Asn50_(LC)-Asn51_(LC) in bothPGT121 and 10-1074, resulting in a less negatively-charged surface. Thelarge number of common substitutions introduced into LC_(PGT121) andLC₁₀₋₁₀₇₄ (approximately 50% of LC substitutions) point to CDRL1, CDRL2and FWR2_(LC) as potential contact regions for epitopes shared by PGT121and 10-1074.

Next, comparisons were made with the structure of PGT128, whichrecognizes Asn332_(gp120)- and Asn301_(gp120)-linked glycans and V3 andwas solved as a complex with an outer domain/mini-V3 loop gp120expressed in cells that cannot produce complex-type N-glycan-modifiedproteins. Unlike the CDRH3 loops of PGT121 and 10-1074, PGT128_(CDRH3)is not tilted towards PGT128_(VL), and CDRH3_(PGT128) does not include atwo-stranded (β-sheet. In addition, CDRH3_(PGT128) (18 residues) isshorter than the CDRH3s of PGT121 and 10-1074 (24 residues), whereasCDRH2_(PGT128) contains a six-residue insertion not found in PGT121 or10-1074. Due to these differences, CDRH2 is the most prominent featurein PGT128, whereas CDRH3 is most prominent in PGT121 and 10-1074.CDRH2_(PGT128) and CDRL3_(PGT128) together recognize Man_(8/9) attachedto Asn332_(gp120), and CDRH3_(PGT128) contacts the V3 loop base. Thismode of gp120 recognition is not possible for PGT121 and 10-1074 becausethe structural characteristics of their CDRH2 and CDRH3 loops differsignificantly from those of PGT128, consistent with the ability ofPGT128, but not PGT121 and 10-1074 (FIG. 7), to recognize protein-freehigh-mannose glycans.

EXAMPLE 7 Crystal Structure of PGT121-Glycan Complex

A 2.4 Å resolution structure of PGT121 associated with a complex-typesialylated bi-antennary glycan was solved (Table 9) using crystalsobtained under conditions including NA2, a complex-type asialylbi-antennary glycan (FIG. 7). Surprisingly, the glycan bound to PGT121in our crystal structure was not NA2, but rather a complex-type N-glycanfrom a neighboring PGT121 Fab in the crystal lattice; specifically theN-glycan attached to Asn105_(HC). The glycan identity is evident becausethere was electron density for the glycosidic linkage to Asn105_(HC) andfor a terminal sialic acid on the Manal -3Man antenna (the galactose andsialic acid moieties of Manal-6Man antenna were unresolved). Thecomposition of the bound glycan corresponds to a portion of theα2-6-sialylated A2(2-6) glycan that was bound by PGT121 in microarrayexperiments (FIG. 7) and to the expected sialyl linkage on complex-typeN-glycans attached to PNGS on proteins expressed in HEK293T cells.Although the V_(H)-V_(L) domains of this structure (“liganded” PGT121)superimpose with no significant differences onto the V_(H)-V_(L) domainsof the PGT121 structure with no bound N-glycan (“unliganded” PGT121)(Table 10), the elbow bend angle (angle between the V_(H)-V_(L) andC_(H)1-C_(L) pseudo-dyads) differs between the structures. Thisdifference likely reflects flexibility that allows the Fab to adoptvariable elbow bend angles depending upon crystal lattice forces.

Given that we observed binding of complex-type N-glycan in one crystalstructure (the “liganded” PGT121 structure) but not in another structure(the “unliganded” PGT121 structure), we estimate that the affinity ofPGT121 for complex-type N-glycan not attached to gp120 is in the rangeof the concentration of PGT121 in crystals (˜10 mM). If we assume thatthe K_(D) for binding isolated glycan is in the range of 1-10 mM,comparable to the 1.6 mM K_(D) derived for PG9 binding toMan₅GlcNAc₂-Asn, then the K_(D) for PGT121 binding of isolated glycanrepresents only a minor contribution to the affinity of PGT121 forgp120, which is in the nM range (FIG. 4A).

The glycan in the “liganded” PGT121 structure interacts exclusively withthe V_(H) domain and makes extensive contacts with residues in all threeCDRs (buried surface area on PGT121_(HC)=600 Å²). Contacts include 10direct and 18 water-mediated hydrogen bonds (Table 11) with 9 aminoacids anchoring the glycan between the N-acetylglucosamine moiety linkedto the branch-point mannose and the terminal sialic acid on the1-3-antenna. Several contacts with PGT121 are made by this sialic acid,including three direct hydrogen bonds with PGT121 residues Asp31_(HC)and His97_(HC) in addition to water-mediated hydrogen bonds withAsp31_(HC). The sialic acid also contributes to a water-mediatedintra-glycan hydrogen bond network. The direct contacts with sialic acidmay explain the stronger binding of PGT121 to the sialylated A2(2-6)glycan than to the asialylated NA2 glycan in our glycan microarrayanalysis (FIG. 7). Extensive water-mediated protein contacts establishedby the N-acetylglucosamine and galactose moieties of the 1-3-antennacould explain the binding observed for asialylated mono- andbi-antennary glycans to PGT121 (FIG. 7).

Six of the residues contributing direct or likely amino acid side chaincontacts to the glycan (Ser32_(HC-CDRH1), Lys53_(HC-CDRH2),Ser54_(HC-CDRH2), Asn58_(HC-CDRH2), His97_(HC-CDRH3),Thr1001_(HC-CDRH3)) differ from those on 10-1074 (Tyr32_(HC-CDRH1),Asp53_(HC-CDRH2), Arg54_(HC-CDRH2), Thr58_(HC-CDRH2), Arg97_(HC-CDRH3),Tyr1001_(HC-CDRH3)), and are highly conserved among PGT121-like, but not10-1074-like, antibodies. The 10-1074 residues lack the correspondingfunctional groups to make the observed glycan contacts or have bulkyside chains that would cause steric clashes. Four of these residues alsodiffer from those on GL (Tyr32_(HC-CDRH1), Tyr53_(HC-CDRH2),Gln97_(HC-CDRH3), Tyr1001_(HC-CDRH3)), suggesting that the lack ofbinding of 10-1074-like antibodies and GL to protein-free complex-typeglycans in our glycan microarrays results from missing hydrogen bondsand/or steric clashes (e.g., His97_(PGT121) versus Arg97₁₀₋₁₀₇₄;Thr1001_(PGT121) versus Tyr1001₁₀₋₁₀₇₄). As the majority of sequencedifferences between PGT121 and 10-1074 cluster in the CDRH loops,specifically to the surface of the cleft between CDRH2 and CDRH3 wherewe observe the bound complex-type N-glycan, differential recognition ofcomplex-type glycans on gp120 may account for some or all of thedifferences in their fine specificity observed.

EXAMPLE 8 Substitution of Glycan-Contacting Antibody Residues AaffectsNeutralization

To evaluate the contributions of complex-type N-glycan contactingresidues identified from the “liganded” PGT121 structure, we generatedtwo mutant antibodies designed to exchange the complex-typeglycan-contacting residues between PGT121 and 10-1074: a 10-1074 IgGwith PGT121 residues (six substitutions in IgH Y32S, D53K, R54S, T58N,R97H, Y1001T) and a PGT121 IgG with reciprocal substitutions. The“glycomutant” antibodies (10-1074_(GM) and PGT121_(GM)) exhibitednear-wildtype apparent affinity for YU-2 gp120/gp140 as measured by SPR(FIG. 2A), demonstrating that the substitutions did not destroy bindingto an envelope spike derived from a viral strain neutralized by bothPGT121 and 10-1074 (FIG. 1A). The fact that PGT121 complex-type N-glycancontacting residues can be accommodated within the 10-1074 backgroundwithout destroying binding to a gp120/gp140 bound by both wildtypeantibodies implies overall similarity in antigen binding despite finespecificity differences.

Unlike wildtype PGT121, PGT121_(GM) showed no glycan binding inmicroarray experiments, confirming that 10-1074 residues at thesubstituted positions are not compatible with protein-free glycanbinding (FIG. 2B) and supporting the suggestion that residues contactingthe glycan in the “liganded” PGT121 structure are involved inrecognition of complex-type glycans in the microarrays. 10-1074_(GM)also showed no binding to protein-free glycans (FIG. 2B), indicating theinvolvement of residues in addition to those substituted in creating thebinding site for a protein-free complex-type N-glycan.

Next, a TZM-b1-based assay was used to compare neutralization of thewildtype and “glycomutant” antibodies. We tested 40 viral strainsincluding strains differentially resistant to PGT121 or 10-1074 andstrains sensitive to both wildtype antibodies (FIG. 2C and Table 12).Consistent with the binding of PGT121_(GM) and 10-1074_(GM) to purifiedYU-2 envelope proteins, both mutants neutralized the YU-2 virus;however, 64% of the PGT121-sensitive strains were resistant toPGT121_(GM) (FIG. 2C, and Table 12) suggesting that theglycan-contacting residues identified in the “liganded” PGT121 structureare relevant to the neutralization activity of PGT121. Conversely,10-1074_(GM) exhibited a higher average potency than wildtype 10-1074against the 10-1074-sensitive strains (FIG. 2C and Table 12), includingpotency increases of >3-fold against four 10-1074-sensitive strains(WITO4160.33, ZM214M.PL15, Cell_H1, and 3817.v2.c59). In general, thePGT121 substitutions into 10-1074 did not confer sensitivity to10-1074_(GM) upon PGT121-sensitive/10-1074-resistant strains, howevertwo of these strains (CNE19 and 62357_14_D3_4589) became sensitive to10-1074_(GM) (IC_(50S)=0.19 μg/ml and 40.8 μg/ml, respectively).Interestingly, these are the only PGT121-sensitive/10-1074-resistantstrains that include an intact Asn33_(gp120)-linked PNGS. The otherPGT121-sensitive/10-1074-resistant strains lack theAsn332_(gp120)-linked glycan and are resistant to PGT121_(GM) and10-1074_(GM), implying that their sensitivity to wildtype PGT121 involvea nearby N-glycan and/or compensation by protein portions of theepitope. Although a dramatic gain of function was observed only for10-1074_(GM) against one strain (CNE19), this result, together with thegeneral improvement observed for 10-1074_(GM) against 10-1074-sensitivestrains (FIG. 2C), is consistent with the interpretation that thecrystallographically-identified glycan-contacting residues can transferPGT121-like recognition properties to 10-1074 in some contexts and/oraffect its potency in others. In addition, the loss of neutralizationactivity for PGT121_(GM) against PGT121-sensitive strains demonstratesthat neutralization activity of PGT121 involves residues identified ascontacting complex-type N-glycan in the “liganded” PGT121 structure.

Results

PGT121 is a glycan-dependent bNAb that was originally identified in theserum of a clade A-infected donor in a functional screen yielding onlytwo clonally-related members. gp140 trimers were used as “bait” forsingle cell sorting to isolate 29 new clonal variants of. The PGT121clonal family includes distinct groups of closely-related antibodies;the PGT121- and 10-1074-groups. The results suggest that the epitopes ofboth groups involve the PNGS at Asn332_(gp120) and the base of the V3loop. The PGT121-like and 10-1074-like antibody groups differ in aminoacid sequences, gp120/gp140 binding affinities, and neutralizingactivities, with the 10-1074-like antibodies being completely dependentfor neutralization upon an intact PNGS at Asn332_(gp120), whereasPGT121-like antibodies were able to neutralize some viral strainslacking the Asn332_(gp120) PNGS.

A notable difference between the two antibody groups is that thePGT121-like antibodies bound complex-type N-glycans in carbohydratearrays, whereas the 10-1074-like antibodies showed no detectable bindingto any of the protein-free N-glycans tested (FIG. 7). Protein-freeglycan binding by anti-HIV antibodies is not always detectable; e.g.,although PG9 recognizes a gp120-associated high-mannose glycan, nobinding to protein-free glycans was detected in microarrays. Thusalthough a positive result in a glycan microarray implies involvement ofa particular glycan in an antibody epitope, a negative result does notrule out glycan recognition. For example, although not detectable in theglycan microarray experiments, high-mannose glycans may be involved inthe PGT121 epitope, consistent with binding and neutralization ofhigh-mannose-only forms of gp120 protein and virions (FIG. 8).

The molecular basis for the differences between PGT121, 10-1074 andtheir GL progenitor was revealed in part by their crystal structures.The finding that the majority of light chain somatic mutations areshared between PGT121 and 10-1074, whereas mutations in the heavy chainsdiffer, suggests that the light chain contacts shared portions of thegp120 epitope and the heavy chain recognizes distinct features. Allthree antibodies exhibit an extended CDRH3 with a non-polar tip that mayallow accessing of cryptic epitopes. Differences in the antigen-bindingsite of the two mature Fabs were mainly localized to a cleft betweenCDRH2 and the extended CDRH3. Interestingly, the putativeantigen-binding cleft between CDRH2 and CDRH3 was also found in arepresentative germline progenitor of PGT121 and 10-1074.

Structural information was obtained concerning glycan recognition byPGT121-like antibodies from a crystal structure in which a complex-typesialylated N-glycan attached to a V_(H) domain residue interacted withthe combining site of a neighboring PGT121 Fab. Several features of the“liganded” PGT121 structure suggest it is relevant for understanding therecognition of complex-type N-glycans on gp120 by PGT121-likeantibodies. First, the glycan in the structure corresponds to the α2-6sialylated glycan A2(2-6) PGT121 binds in microarrays (FIG. 7). Second,the glycan interacts with PGT121 using the cleft between CDRH3 and CDRH2that was suggested by structural analyses to be involved in epitoperecognition, potentially explaining the unusual tilting of CDRH3 towardsVL in the PGT121 and 10-1074 structures. Third, most of the V_(H)residues identified as interacting with the glycan differ between PGT121and 10-1074, rationalizing different binding profiles in glycanmicroarrays and potentially explaining the different fine specificitiesrevealed in protein binding experiments. Fourth, swappingcrystallographically-identified glycan contact residues between PGT121and 10-1074 in part transferred their properties: PGT121_(GM), like10-1074, did not bind to protein-free glycans, but both PGT121_(GM) and10-1074_(GM) preserved near wildtype binding to purified YU-2gp120/gp140. Although PGT121_(GM) retained the ability to neutralizesome viral strains that were neutralized by wildtype PGT121 and 10-1074,it failed to neutralize strains that arePGT121-sensitive/10-1074-resistant, demonstrating that theglycan-binding motif is essential for the neutralizing activity ofPGT121 against 10-1074-resistant strains. For the reciprocal swap, theneutralization potency of 10-1074_(GM) was increased or unaffectedrelative to 10-1074, and in one case, 10-1074_(GM) potently neutralizeda PGT121-sensitive/10-1074-resistant strain, consistent with transfer ofthe crystallographically-identified glycan motif and the hypothesis thatthe epitopes of PGT121- and 10-1074-like antibodies are related. Inanalyses of gp120 sequences from strains for which PGT121 neutralizationdata are available, other than a correlation with the PNGS atAsn332gp120 for viruses sensitive to PGT121-like and 10-1074-likeantibodies, no clear pattern of PNGS usage emerges for the differentcategories of viral strains (PGT121-sensitive/10-1074-sensitive,PGT121-sensitive/10-1074-resistant, PGT121-resistant/10-1074-sensitive)except that the 10-1074-resistant strains generally lack theAsn332gp120-associated PNGS.

EXAMPLE 9 Passive Transfer of Anti-HIV-1 Neutralizing mAbs In-Vivo

Five isolated potent and broadly acting anti-HIV neutralizing monoclonalantibodies were administered to rhesus macaques and challenged themintrarectally 24 h later with either of two different SHIVs. Bycombining the results obtained from 60 challenged animals, theprotective neutralization titer in plasma preventing virus acquisitionin 50% of the exposed monkeys was approximately 1:100.

Animal Experiments

The macaques used in this study were negative for the MEC class IMamu-A*01 allele.

Construction of the R5-tropic SHIVDH12-V3AD8

PCR mutagenesis, with primers corresponding to the 5′ and 3′ halves ofthe SHIVAD8EO (PNAS 109, 19769-19774 (2012)) gp120 V3 coding region(forward primer:AGAGCATTTTATACAACAGGAGACATAATAGGAGATATAAGACAAGCACATTGCAACATTAGTAAAGTAAAATGGC (SEQ ID NO: 214) and reverse primer:TCCTGGTCCTATATGTATACTTTTCCTTGTATTGTTGTTGGGTCTTGTACAATTAATTTCTACAGTTTCATTC (SEQ ID NO: 215)), was employed to introduce these V3sequences into the genetic background of the pSHIVDH12.CL7 molecularclone (J. of Virology 78, 5513-5519 (2004)), using Platinum PFX DNApolymerase (Invitrogen). Following gel purification, the PCR product wastreated with T4 polynucleotide kinase (GibcoBRL) and blunt-end ligatedto create pSHIVDH12.V3AD8, which was used to transform competent cells.

Viruses

Virus stocks were prepared by first transfecting 293T cells with theSHIVAD8EO or SHIVDH12-V3AD8 molecular clones using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.). Culture supernatants were collected 48 hlater and aliquots stored at −80° C. until use. ConcanavalinA-stimulated rhesus PBMCs (2×10⁶ cells in 500 μl) were infected withtransfected cell supernatants by spinoculation (J. of Virology 74,10074-10080 (2000)) for 1 h, mixed with the same number/volume ofactivated PBMC, and cultures were maintained for at least 12 days withdaily replacement of culture medium. Samples of supernatant medium werepooled around the times of peak RT production to prepare individualvirus stocks.

Antibodies

Eleven monoclonal antibodies (VRC01, NIH45-46, 45-46G54W, 45-46m2,3BNC117, 12A12, 1NC9, and 8ANC195, 10-1074, PGT121, and PGT126) wereisolated and produced. DEN3, a dengue virus NS1-specific human IgG1monoclonal antibody (PNAS 109, 18921-18925 (2012)), or control human IgG(NIH Nonhuman Primate Reagent Resource) were used as the negativecontrol antibodies in this study. The monoclonal antibodies selected forpre-exposure passive transfer were administered intravenously 24 hbefore virus challenge.

Quantitation of plasma viral RNA levels.

Viral RNA levels in plasma were determined by real-time reversetranscription-PCR (ABI Prism 7900HT sequence detection system; AppliedBiosystems).

Antibody concentrations in plasma.

The concentrations of administered monoclonal antibodies in monkeyplasma were determined by enzyme-linked immunosorbent assay (ELISA)using recombinant HIV-1JRFL gp120 (Progenics Pharmaceuticals) or HIVIIIB(Advanced Biotechnology inc) (J. of Virology 75, 8340-8347 (2001)).Briefly, microtiter plates were coated with HIV-1 gp120 (2 μg/ml) andincubated overnight at 4° C. The plates were washed with PBS/0.05%Tween-20 and blocked with 1% (vol/vol) BSA. After blocking, serialdilution of antibodies or plasma samples were added to the plate andincubated for 1 h at room temperature. Binding was detected with a goatanti-human IgG F(ab)2 fragments coupled to alkaline phosphatase (Pierce)and visualized with SIGMAFAST OPD (Sigma-Aldrich). The decay half-livesof neutralizing monoclonal antibodies were calculated by asingle-exponential decay formula based on the plasma concentrationsbeginning on day 5 or day 7 post antibody administration (J. of Virology84, 1302-1313 (2010)).

Neutralization assays.

The in vitro potency of each mAb and the neutralization activity presentin plasma samples collected from rhesus macaques were assessed by twotypes of neutralization assays; 1) TZM-b1 entry assay with pseudotypedchallenge virus (AIDS Res Hum Retroviruses 26, 89-98 (2010)) or 2) a 14day PBMC replication assay with replication competent virus (J. ofvirology 76, 2123-2130 (2002)). For the TZM-b1 assay, serially dilutedmAb or plasma samples were incubated with pseudotyped viruses,expressing env gene derived from SHIVAD8EO or SHIVDH12.V3AD8 andprepared by cotransfecting 293T cells with pNLenv1 and pCMV vectorsexpressing the respective envelope proteins (J. of Virology 84,4769-4781 (2010)). The 50% neutralization inhibitory dose (IC50) titerwas calculated as the dilution causing a 50% reduction in relativeluminescence units (RLU) compared with levels in virus control wellsafter subtraction of cell control RLU (J. of Virology 84, 1439-1452(2010)). The neutralization phenotype (tier levels) of theSHIVDH12.V3AD8 molecular clone was determined by TZM-b1 cell assay usingplasma samples from a cohort study, which exhibit a wide range ofneutralizing activities against subtype B HIV-1 isolates (J. of GeneralVirology 91, 2794-2803 (2010)).

Determinations of animal protective titers and statistical analyses.

Calculation of the neutralizing titer in plasma against each R5 SHIV,resulting in the prevention of virus acquisition of 50 or 80% of thevirus-challenged animals, was performed using the method of Reed andMuench (Am J Hyg 27, 493-497 (1938)). One significant outlier animal(DEW7) was omitted from the calculation. Probit regression was used tomodel the relationship between the titers in plasma required to confersterilizing immunity in vivo using all 60 passively immunized monkeys(Cambridge University Press, Cambridge, England, ed. 3rd, 2007), withp-values from this model based on Likelihood ratio Tests. Plasma titersneeded for different levels of in vivo protection (33%, 50%, 80%, 90%,and 95%) were determined from the probit model estimates and the methodof bootstrapping was used to construct 90% confidence intervals.Results:

SHIVDH12-V3AD8, like SHIVAD8EO, possesses Tier 2 anti-HIV-1neutralization sensitivity properties (Table 13). Rhesus macaquesinoculated intravenously or intrarectally with SHIVDH12-V3AD8 exhibitedpeak viremia ranging from 105 to 107 viral RNA copes/ml of plasma atweeks 2 to 3 post infection (PI). In most SHIVDH12-V3AD8 infectedanimals, plasma viral loads decline to background levels between weeks 8to 20 PI.

The neutralization sensitivity of SHIVAD8EO to 11 recently reportedbroadly reacting anti-HIV-1 mAbs was initially determined in the TZM-b1assay system (FIGS. 11A and B). Eight of these antibodies, VRC01,NIH45-46 (23), 45-46G54W, 45-46m2, 3BNC117, 12A12, 1NC9, and 8ANC195targeted the gp120 CD4 bs (Science 333, 1633-1637 (2011)) and three,10-1074, PGT121, and PGT126 (Nature 477, 466-470 (2011)), were dependenton the presence of the HIV-1 gp120 N332 glycan. When tested againstSHIVAD8EO, all three glycan-dependent mAbs exhibited greater potencythan the CD4 bs mAbs (FIG. 11A). The IC50 values for the three mAbstargeting the gp120 N332 glycan ranged from 0.09 to 0.15 μg/ml. The CD4bs mAbs exhibited a much broader range (0.14 to 6.34 μg/ml) of IC50neutralizing activity with 3BNC117 being the most potent. A similarhierarchy (glycan-dependent>CD4 bs dependent) of neutralizing mAbpotency was also observed with SHIVDH12-V3AD8, but the neutralizingactivity was distributed across a much wider (>100 fold) range comparedto the IC50 values observed for SHIVAD8EO (FIG. 11B). SHIVDH12-V3AD8 wassomewhat more sensitive to the glycan targeting mAbs and more resistantto the CD4 bs neutralizing mAbs than SHIVAD8EO.

Based on the results shown in FIG. 11, five neutralizing mAbs wereselected for a pre-exposure passive transfer study: VRC01, because itwas the first CD4bs NAb of the newly isolated broadly acting NAbs to becharacterized; the CD4 bs mAbs 45-46m2 and 3BNC117, both of whichexhibited strong neutralizing activity against SHIVAD8EO andSHIVDH12-V3AD8; and the gp120 N332 glycan-dependent mAbs, PGT121 and10-1074.

The protocol for passive transfer experiments was to administerdecreasing amounts of neutralizing mAbs intravenously and challengeanimals intrarectally 24 h later. The goal was to block virusacquisition, coupled with the knowledge that repeated administrations ofhumanized anti-HIV mAbs to individual macaques could reduce theirpotency and/or possibly induce anaphylactic responses, a SHIV challengedose of sufficient size to establish an in vivo infection following asingle inoculation was chosen. In this regard, we had previouslyconducted intrarectal titrations of SHIVAD8 in rhesus monkeys andreported that the inoculation of 1×103 TCID50, determined by endpointdilution in rhesus macaque PBMC, was equivalent to administeringapproximately 3 animal infectious doses50 (AID50) (J. of virology 86,8516-8526 (2012)). In fact, single intrarectal inoculations of 3 AID50have resulted in the successful establishment of infection in 10 of 10rhesus macaques with SHIVAD8EO or SHIVDH12-V3AD8.

As a control for the first passive transfer experiment, an anti-denguevirus NS1 IgG1 mAb was administered intravenously to animals, which werechallenged with SHIVAD8EO 24 h later. Both monkeys (ML1 and MAA) rapidlybecame infected, generating peak levels of plasma viremia at week 2 PI.VRC01 was the first anti-HIV-1 neutralizing mAb tested for protectionagainst virus acquisition and was administered to two macaques at a doseof 50 mg/kg. One (DEGF) of the two inoculated macaques was completelyprotected from the SHIVAD8EO challenge, with no evidence of plasmaviremia or cell-associated viral DNA over a 45 week observation period.The other recipient of 50 mg/kg VRC01 (DEH3) became infected, but peakplasma viremia was delayed until week 5 PI. Two additional macaquesadministered lower amounts (20 mg/kg) of VRC01 were not protected fromthe SHIVAD8EO challenge. These results are summarized in Table 13.

Examined next, the protective properties of PGT121 against a SHIVAD8EOchallenge. PGT121 was one of the most potent glycan targetingneutralizing mAbs measured in the TZM-b1 assay (FIG. 11). Based on theresults obtained with VRC01, in vivo PGT121 mAb titration at 20 mg/kgwas chosen to begin with. The two challenged monkeys (KNX and MK4)resisted the SHIVAD8EO challenge. When lower amounts (viz. 5 mg/kg, 1mg/kg, or 0.2 mg/kg) of PGT121 were administered, 1 of 2, 2 of 2, and 0of 2 animals, respectively, were protected (Table 13).

The capacity of VRC01 and PGT121 mAbs to block SHIVDH12-V3AD8acquisition was similarly evaluated (Table 13). The results obtainedwith VRC01 were comparable to those observed with the SHIVAD8EOchallenge: 1 of 2 recipients of 30 mg/kg was protected from theestablishment of a SHIVDH12-V3AD8 infection. The PGT121 mAb wasconsiderably more potent than VRC01 in preventing SHIVDH12-V3AD8acquisition: 2 of 2 recipients of 0.2 mg/kg PGT121 resisted infection.PGT121 also appeared to be somewhat more effective in preventingSHIVDH12-V3AD8 versus SHIVAD8EO in vivo infections (Table 13). Thisresult is consistent with the 8-fold difference in IC50 values forPGT121 for neutralizing the two SHIVs in in vitro assays (FIG. 11).

The results of passively transferring 10-1074, 3BNC117, or 45-46m2neutralizing mAbs to rhesus monkeys, followed by a challenge with eitherSHIVAD8EO or SHIVDH12-V3AD8, are summarized in Table 13. The 10-1074 mAbpotently blocked the in vivo acquisition of both SHIVs. The CD4bs3BNC117 and 45-46m2 mAbs were selected for passive transfer to macaquesbased on their IC50 values against both SHIVs in the in vitroneutralization experiments shown in FIG. 11. 3BNC117 successfullyblocked SHIVAD8EO infection in 2 of 2 monkeys at 5 mg/kg but not in 2other animals given a dose of 1 mg/kg (Table 13). This was similar tothe results observed when the same amounts of 3BNC117 were administeredto macaques challenged with SHIVDH12-V3AD8: 1 of 2 became infected at 5mg/kg; 1 of 2 became infected at 1 mg/kg.

Plasma samples collected at various times from passively transferredmacaques were analyzed by HIV-1 gp120 ELISA to determine neutralizingmAb concentrations. In general, the plasma concentrations of each mAb atthe time of challenge (24 h following antibody administration)correlated with the dose of antibody administered (Table 13).

The relationships of plasma mAb concentrations to in vivo protection areshown in FIG. 12. Of the 5 neutralizing mAbs evaluated, PGT121 wasclearly the most effective against both viruses, with SHIVDH12-V3AD8exhibiting somewhat greater sensitivity to this mAb (2 of 2 monkeysprotected at a plasma concentration of 0.2 μg/ml). In contrast, a plasmaconcentration of nearly 400 μg/ml of VRC01 was required to protect 1 of2 animals against the same SHIVDH12-V3AD8 challenge virus (Table 13).The most potent CD4 bs mAb administered to macaques in this study,3BNC117, was approximately 6 to 10-fold more effective than VRC01 inpreventing the acquisition of either SHIV (FIG. 12, Table 13).

The calculated half lives of PGT121, 10-1074, 3BNC117, and VRC01 mAbswere quite similar: 3.5days, 3.5 days, 3.3 days, and 3.1 days,respectively. In contrast, the half-life of 45-46m2 was extremely shortand could not be determined. Based on the plasma mAb concentrations inseveral macaques 24 h following the administration of 20 mg/kg ofhumanized neutralizing mAbs (viz. approximately 250 μg/ml [Table 13]),the two monkeys receiving 20 mg/kg of 45-46m2 had plasma mAbconcentrations of only 15.0 and 17.6 μg/ml, a decay of more than 95%relative to other neutralizing mAbs in 24 h.

Neutralization titers were measured on plasma samples collected 24 hfollowing mAb administration when the macaques were challenged withSHIVAD8EO or SHIVDH12-V3AD8. As shown in Table 13, good correlation wasobserved between anti-viral plasma neutralization titers and protectionfrom SHIV infection. The administration of the two glycan-dependent mAbs(PGT121 and 10-1074) clearly resulted in the highest titers ofanti-HIV-1 neutralizing activity at the time of virus challenge. Thetiters measured in recipients of the 45-46m2 mAb were at the limits ofdetection or undetectable due to its extremely short half-life in vivo.

The method described by Reed and Muench (Am J Hyg 27, 493-497 (1938))was used to calculate the neutralization titers, measured in plasma,needed to prevent virus acquisition in 50% of challenged monkeys. Theseprotective titers for the 28 monkeys, challenged with SHIVAD8EO, or the32 monkeys, challenged with SHIVDH12-V3AD8, were separately deduced(Tables 15 and 16). The plasma neutralization titers required forprotecting 50% of the SHIVAD8EO or SHIVDH12-V3AD8 challenged animalswere calculated to be 1:115 and 1:96, respectively. Because thesesimilar titers were obtained following: 1) SHIV challenges by identicalroutes and inoculum size and 2) the administration of the same ensembleof neutralizing mAbs, the neutralization data from all 60 animals werecombined and subjected to probit regression to examine the relationshipbetween plasma neutralization titers and in vivo protection. As afurther check, when a term for the SHIV virus was included in the probitregression model on all 60 macaques, there was no evidence of adifference between the two SHIV viruses (p=0.16). When applied to theentire group of 60 macaques, probit regression estimated that plasmaneutralization titers of 1:104 would prevent virus acquisition in 50% ofanimals. Probit analysis of the data also estimates that 50% plasmaneutralization titers of 1:57 or 1:329 would protect 33% or 80%,respectively, of exposed animals.

EXAMPLE 10 Administration of Neutralizing mAbs to Chronically InfectedHIV In-Vivo Models

Methods Summary: The neutralization activities of the broadly acting3BNC11724 and 10-107423 neutralizing mAbs against SHIVAD8EO wereinitially determined in the TZM-b1 cell system against SHIVAD8EO. Theircapacities to block virus acquisition or to control plasma viremia inchronically infected animals challenged with the R5-tropic SHIVAD8EOwere assessed by monitoring plasma viral loads and cell-associated viralnucleic acids; levels of CD4+ T cell subsets were measured by flowcytometry. SGA analyses of circulating viral variants and thedetermination of antibody levels in plasma. Plasma concentration of NAbswas determined by measuring neutralizing activity against HIV-1pseudovirus preparations only susceptible to either 10-1074 or 3BNC117.

Results:

Two groups of chronically infected macaques were assessed. The firstgroup consisted of two clinically asymptomatic animals (DBZ3 and DC99A)that had been infected for 159 weeks and had sustained similar andsignificant declines of circulating CD4+ T cells (Table 17). The regimenfor treating ongoing SHIV infections was to co-administer 101074 and3BNC117, at a dose of 10 mg/kg. At the time of mAb administration, theplasma viral loads in macaques DBZ3 and DC99A were 1.08×104 and 7.6×103RNA copies/ml, respectively. Both monkeys responded to combinationanti-HIV-1 mAb treatment with immediate and rapid reductions of plasmaviremia to undetectable levels within 7 to 10 days. Suppression ofmeasurable SHIVAD8EO in the plasma of macaques DBZ3 and DC99A, followinga single administration of the two mAbs, lasted 27 and 41 days,respectively. In each case, plasma viremia rebounded to pretreatmentlevels.

A second group of three animals (DBX3, DCF1, and DCM8), each of whichwere also infected with SHIVAD8EO for more than 3 years and wereclinically symptomatic with intermittent diarrhea and or anorexia, weretreated with the two neutralizing antibodies (Table 17). At the time ofmAb administration, the level of circulating CD4+ T cells in macaqueDCM8 was only 43 cells/μl and somewhat higher in animals DCF1 (105cells/μl) and DBXE (158 cells/μl). Plasma viral loads exceeded 105 RNAcopies/ml in animals DBXE and DCF1 and were significantly lower(1.59×103 RNA copies/ml) in monkey DCM8. The administration of the twomAbs to monkey DBXE resulted in a biphasic reduction of viremia from2.0×105 RNA copies at day 0 to undetectable levels in plasma at day 20.This was followed, within a few days, by a resurgence of high levels ofcirculating virus in DBXE. Macaque DCM8, with more modest plasma virusloads and very low numbers of circulating CD4+ T cells, experienced arapid decline of viremia to undetectable levels between days 6 and 20following the initiation of mAb treatment. Finally, animal DCF1,previously reported to have generated broadly reacting anti-HIV-1 NAbs,exhibited a transient and a comparatively modest 27-fold reduction ofplasma viremia by day 6 in response to combination mAb therapy, beforethe viral loads returned to high pretreatment levels.

PBMC associated viral RNA and DNA levels were also determined prior toand following antibody administration (Table 18). For each animal, mAbtreatment resulted in reduced levels of cell associated viral RNA,correlating well with the plasma viral load measurements. No consistentpattern was observed for cell associated viral DNA levels as a result ofantibody treatment. Administration of neutralizing mAbs to chronicallySHIVAD8EO infected monkeys also had beneficial effects on circulatingCD4+ T cell levels, particularly in animals with very high virus loads.The CD4+ T cell numbers in macaques DBXE and DCF1 increased 2 to 3 foldduring the period of mAb mediated virus suppression, but graduallydeclined to pretreatment levels as viremia again became detectable.

Plasma concentrations of each mAb were determined by measuring theplasma neutralizing activity against selected HIV-1 pseudovirus strainssensitive to one or the other, but not to both antibodies (FIG. 13A). Inevery treated animal, suppression of SHIVAD8EO viremia was maintaineduntil a threshold plasma mAb concentration of approximately 1 to 3 μg/mlwas reached (FIGS. 13B and 13C). This was even the case for macaqueDCF1, for which a modest and transient reduction of plasma viral RNAlevels was observed. Interestingly, the mAbs administered to clinicallysymptomatic macaques DCM8 and DCF1 had shortened half-lives or wereundetectable. As noted earlier, macaque DCM8 had extremely low CD4+ Tcell levels (43 cells/μl plasma) and macaque DCF1 had to be euthanizedon day 56 post treatment initiation due to its deteriorating clinicalcondition. A necropsy of DCF1 revealed severe enteropathy, characterizedby disseminated gastrointestinal cryptosporidiosis, pancreatitis, andcholangitis.

SGA analysis was used to determine whether amino acid substitutions hadarisen in gp120 regions previously shown to affect the sensitivity to10-1074 or 3BNC117 mAbs. In each case the rebound virus present inplasma following immunotherapy was unchanged. To further test thesensitivity of the re-emerging viruses, 10-1074 plus 3BNC117 combinationtherapy (10 mg/kg of each) was re-administered to the two clinicallyasymptomatic monkeys (DBZ3 and DC99A). The viral loads in each animalagain rapidly fell, becoming undetectable at day 7 of the secondimmunotherapy cycle. Viremia was suppressed for 7 days in macaque DBZ3and more than 21 days in monkey DC99A. Taken together, these resultssuggest that the re-emergence of virus following the first treatmentcycle in these two animals represented insufficient mAb levels in vivorather than antibody selected virus resistance.

TABLE 3 Repertoire of PGT121 and 10-1074 clonal variants pt10 FRW1_FRW3_ mAB# VH DH JH CD43¹ VHmut Length¹ (-) (+) Y Lc Vλ Jλ LCDR3¹ Vλmutdel Ins Length¹ (-) (+) Y 10-160 4-59 3-3/16 6 ARRGQRIYWVVSFGEFFYYYSMDV49 24 2 3 4 / / / / / / / / / 10-186 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 52 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 47 12 3 12 12 0 10-248 4-59 3-3/16 6 ARRGQRIYGVVSSGEFFYYYSMDV 46 24 2 3 4 / / / /10-295* 4-59 / 6 TKHGRRIYGIVAFNEWFTYFYMDV 63 24 2 4 3 λ 3-21 3HIYDARGGTNWV 58 21 3 12 1 2 1 10-266 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 12 0 10-267 4-59 / 6 AQQGKRIYGIVSFGELFYYYMDA 58 24 2 2 5 / / / / 10-303*4-59 3-3/9 6 TLHGRRIYGIVAFNEWFTYFYMDV 54 24 2 3 3 λ 3-21 3 HIWDSRVPTKWV50 21 3 12 1 3 0 10-354 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 48 24 2 34 / / / / 10-410* 4-59 3-10/3 6 ALHGKRIYGIVALGELFTYFYMDV 63 24 2 3 3 λ3-21 3 HIWDSRRPTNWV 44 21 3 12 1 3 0 10-416 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 12 0 10-468 4-59 3-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3HMWDSRSGFSWS 46 12 3 12 1 2 0 10-543 4-59 3-10/3 6ALHGKRIYGIVALGELFTYFYMDV 60 24 2 3 3 / / / / / / / / / 10-570 4-593-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 42 24 2 3 4 / / / / / / / / / 10-6214-59 3-3/9 6 TLHGRRIYGIVAFNEWFTYFYMDV 54 24 2 3 3 / / / / / / / / /10-664 4-59 3-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 47 24 2 3 4 / / / / / / // / 10-720 4-59 3-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 / / / / // / / / 10-730 4-59 3-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 48 24 2 3 4 / / // / / / / / 10-814 4-59 3-10 6 TQQGKRIYGVVSFGEFFHYYYMDA 43 24 2 3 4 λ3-21 3 HKWDSRSPLSWV 52 15 3 12 1 3 0 10-847* 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 12 0 10-848 4-59 3-3/9 6 TLHGRRIYGIVAFNEWFTYFYMDV 55 24 2 3 3 λ 3-21 3HIWDSRVPTKWV 46 21 3 12 1 3 0 10-996 4-59 3-3/10 6TQQGKRIYGVVSFGEFFHYYYMDA 41 24 2 3 4 λ 3-21 3 HKWDSRSPLSWV 50 15 3 12 12 0 10-1022 4-59 3-3 6 ARRGQRIYGVVSFGEFFYYYSMDV 51 24 2 3 4 / / / / / // / / 10-1059 4-59 3-3/16 6 TKHGRRIYGVVAFNEWFTYFYMDV 59 24 2 4 3 λ 3-213 HIYDARRPTNWV 46 21 3 12 1 3 1 10- 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 12 0 1074* 10- 4-59 3-10/ 6 ALHGKRIYGIVALGELFTYFYMDV 63 24 2 3 3 λ 3-21 3HIWDSRRPTNWV 44 21 3 12 1 3 0 1121* 3-3 10- 4-59 3-10/ 6ALHGKRIYGIVALGELFTYFYMDV 60 24 2 3 3 λ 3-21 3 HIWDSRRPTNWV 42 21 3 12 13 0 1130* 3-3 10- 4-59 3-10/ 6 ALHGKRIYGIVALGELFTYFYMDV 63 24 2 3 3 λ3-21 3 HIWDSRRPTNWV 45 21 3 12 1 3 0 1141* 3-3 10- 4-59 3-10 6AQQGKRIYGIVSFGEFFYYYMDA 58 24 2 2 5 λ 3-21 3 HYWDSRSPISWV 61 15 3 12 1 21 1145* 10-1151 4-59 3-3 6 ARRGQRIYGVVSFGEFFYYYSMDV 48 24 2 3 4 λ 3-21 3HMWDSRSGFSWS 46 12 3 12 1 2 0 10-1167 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 / / 3 / / / / / / 10-1223 4-593-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 47 24 2 3 4 / / / / / / / / / 10-12324-59 3-10/ 6 ALHGKRIYGIVALGEFLTYFYMDV 58 24 2 3 3 / 3-21 / / / / / / /3-3 10-1263 4-59 3-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3HMWDSRSGFSWS 46 12 3 12 1 2 0 10-1294 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 12 0 10- 4-59 3-3/16 6 ARRGQRIYGVVSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3HMWDSRSGFSWS 45 12 3 12 1 2 0 1341* 10-1342 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 48 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 12 0 10- 4-59 3-3/16 6 TKHGRRIYGVVAFGEWFTYFYMDV 57 24 2 4 3 λ 3-21 3HIYDARRPTNWV 43 21 3 12 1 3 1 1369* 10-1476 4-59 3-3/16 6ARRGQRIYGVVSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 12 0 VHmut and Vλmut indicate the total mumber of mutations in the VH andVL Ig genes. (-) and (+) indicate the numbers of negatively andpositively charged ammino acids in the Ig complementary determiningregion (CDR3), respectively. Y indicated the number of Tyrosine residuesin the IgH/L CDR3s. ¹Based on Kabat nomenclature (IgBLAST). FRW1_del,number of deleted nucleotides in framework region 1 (FRW1) of the IgL.FRW3_ins, number of inserted nucleotides in framework region 3 (FRW3) ofthe IgL. Clonal members with identical IgH sequences are indicated andamong them, IgL sequence identity that defines clones. *indicates therepresentative antibody variants that were produced and analyzed. 10-266IgL was not cloned, and 10-1141 IgG was not produced.

TABLE 4 In vitro TZM-bl neutralization assay on the basic pane 10-136910-259 PGT121 10-303 10-410 10-1130 10-1121 10-1146 10-996 10-134110-847 10-1074 IC50 BaL.26 0.069 0.021 0.021 0.045 0.016 0.013 0.0460.064 0.045 0.032 0.022 0.033 SS1196.1 0.033 0.012 0.008 0.015 0.0080.008 0.029 0.027 0.007 0.011 0.006 0.010 6535.3 0.023 0.005 0.007 0.0140.003 0.003 0.008 0.022 0.018 0.009 0.011 0.007 QH0692.42 0.503 0.1551.085 3.122 2.630 4.871 4.187 0.590 0.395 0.335 0.259 0.259 TRJO4551.580.569 0.189 3.896 14.401 18.511 36.880 15.360 0.548 0.516 0.333 0.2100.170 SC422661.8 0.195 0.096 0.263 0.333 0.132 0.070 0.173 0.195 0.2550.189 0.137 0.145 PVO.4 0.225 0.175 0.147 0.670 0.494 0.385 0.570 0.3100.211 0.236 0.172 0.178 CAAN5342.A2 0.070 0.020 0.013 0.020 0.012 0.0090.033 0.032 0.007 0.009 0.006 0.007 YU-2 0.210 0.135 0.098 0.190 0.0890.078 0.152 0.275 0.256 0.234 0.161 0.143R1166.c1 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40MuLV >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 IC80 BaL.26 0.2680.101 0.081 0.156 0.066 0.062 0.154 0.203 0.228 0.159 0.112 0.124SS1196.1 0.033 0.037 0.030 0.055 0.030 0.037 0.098 0.073 0.040 0.0400.026 0.127 6535.3 0.060 0.022 0.041 0.053 0.021 0.013 0.033 0.078 0.0850.038 0.044 0.044 QH0692.42 1.714 0.551 14.976 18.122 12.071 >40 21.9431.993 1.404 1.100 0.908 0.861 TRJO4551.58 3.818 0.96526.930 >40 >40 >40 >23 2.604 4.265 1.226 0.768 0.693 SC422661.8 0.9400.333 0.714 1.156 0.449 0.264 0.741 0.663 0.845 0.501 0.386 0.392 PVO.40.787 0.716 1.097 2.199 1.572 1.783 2.465 1.319 1.715 0.754 0.774 0.766CAAN5342.A2 0.186 0.063 0.056 0.092 0.055 0.045 0.095 0.088 0.060 0.0540.035 0.044 YU-2 0.738 0.382 0.356 0.502 0.243 0.313 0.340 0.750 0.8910.766 0.537 0.398R1166.c1 >40 >40 >40 >40 >40 >40 >23 >40 >40 >40 >40 >40MuLV >40 >40 >40 >40 >40 >40 >23 >40 >40 >40 >40 >40 Numbers indicateantibody lgG concentrations in μg/ml to reach the IC₅₀ (top) and IC₈₀(bottom) in the TZM-bl neutralization assay. IC_(50/80) values areindicated. >indicates that the IC₅₀ for a given virus was not reached atthe concentration tested. Murine leukemia virus (MuLV) and R1166.c1(clade AE) are negative controls.

TABLE 5 In vitro TZM-bl neutralization assay on the extended panel -IC50 values Virus ID Clade 10-996 10-1074 PGT121 6535.3 B 0.017 0.0140.008 QH0692.42 B 0.396 0.191 1.041 SC422661.8 B 0.173 0.091 0.101 PVO.4B 0.186 0.074 0.131 TR0.11 B 0.012 0.008 0.005 AC10.0.29 B 0.067 0.0220.037 RHPA4259.7 B 0.034 0.021 0.014 THRO4156.18 B >50 >50 >50REJO4541.67 B >50 >50 3.607 TRJO4551.58 B 0.147 0.170 3.728 WITO4160.33B 0.538 0.185 0.459 CAAN5342.A2 B 0.013 0.007 0.011 YU-2 B 0.256 0.1430.098 WEAU_d15_410_787 B(T/F) 0.147 0.104 0.083 1006_11_C3_1601 B(T/F)0.001 0.003 0.008 1054_07_TC4_1499 B(T/F) 0.260 0.129 0.1151056_10_TA11_1826 B(T/F) 0.117 0.038 0.066 1012_11_TC21_3257 B(T/F)0.018 0.008 0.008 6240_08_TA5_4622 B(T/F) 0.095 0.068 0.1286244_13_B5_4576 B(T/F) 0.353 0.202 0.249 62357_14_D3_4589 B(T/F)23.300 >50 1.036 SC05_8C11_2344 B(T/F) 0.069 0.052 0.093 Du156.12 C0.018 0.015 0.007 Du172.17 C 0.173 0.121 0.115 Du422.1 C 0.056 0.0450.029 ZM197M.PB7 C >50 >50 >50 ZM214M.PL15 C 0.413 0.174 0.236ZM233M.PB6 C 0.060 1.451 ZM249M.PL1 C >50 >50 >50 ZM53M.PB12 C >50 >50<0.001 ZM109F.PB4 C >50 >50 7.894 ZM135M.PL10a C 0.099 0.069 0.576CAP45.2.00.G3 C >50 >50 0.086 CAP210.2.00.E8 C 24.793 >50 5.082HIV-001428-2.42 C 0.040 0.044 0.028 HIV-0013095-2.11 C 31.531 >50 >50HIV-16055-2.3 C >50 >50 0.444 HIV-16845-2.22 C 1.325 1.169 12.685Ce1086_B2 C(T/F) >50 >50 <0.001 Ce0393_C3 C(T/F) >50 >50 >50 Ce1176_A3C(T/F) 0.043 0.018 0.017 Ce2010_F5 C(T/F) >50 >50 >50 Ce0682_E4C(T/F) >50 >50 >50 Ce1172_H1 C(T/F) 0.058 0.047 0.023 Ce2060_G9C(T/F) >50 >50 >50 Ce703010054_2A2 C(T/F) >50 >50 >50 BF1266.431aC(T/F) >50 >50 >50 246F C1G C(T/F) 0.092 0.022 0.083 249M B10C(T/F) >50 >50 >50 ZM247v1(Rev-) C(T/F) 0.055 0.042 0.0277030102001E5(Rev-) C(T/F) 0.013 0.006 0.010 1394C9G1(Rev-) C(T/F) 0.0860.050 0.486 Ce704809221_1B3 C(T/F) 0.243 0.139 0.098 CNE19 BC 3.45250.000 0.018 CNE20 BC <0.001 <0.001 0.002 CNE21 BC 0.086 0.087 0.020CNE17 BC 4.040 2.686 45.289 CNE30 BC 0.614 0.363 0.101 CNE52 BC 4.5251.226 3.741 CNE53 BC 0.057 0.039 0.055 CNE58 BC 0.570 0.267 >50 MS208.A1A >50 >50 >50 Q23.17 A 0.008 0.006 0.010 Q451.e2 A >50 >50 >50 Q769.d22A >50 >50 >50 Q259.d2.17 A >50 >50 8.990 Q842.d12 A >50 >50 0.0233415.v1.c1 A 35.876 >50 >50 3365.v2.c2 A 0.286 0.131 0.921 0260.v5.c36 A0.160 0.099 0.054 191955_A11 A (T/F) >50 >50 >50 191084 B7-19 A (T/F)0.057 0.032 0.042 9004SS_A3_4 A (T/F) 0.012 0.011 0.008 T257-31CRF02_AG >50 >50 >50 928-28 CRF02_AG 1.331 0.847 >50 263-8 CRF02_AG10.919 0.666 3.347 T250-4 CRF02_AG <0.001 <0.001 0.001 T251-18 CRF02_AG0.939 1.081 >50 T278-50 CRF02_AG 14.010 2.146 >50 T255-34 CRF02_AG28.369 >50 6.725 211-9 CRF02_AG 0.750 0.112 1.455 235-47 CRF02_AG 0.1280.050 0.332 620345.c01 CRF01_AE >50 >50 >50 CNE8 CRF01_AE >50 >50 >50C1080.c03 CRF01_AE >50 >50 >50 R2184.c04 CRF01_AE >50 >50 >50 R1166.c01CRF01_AE >50 >50 >50 C2101.c01 CRF01_AE >50 >50 >50 C3347.c11CRF01_AE >50 >50 >50 C4118.c09 CRF01_AE >50 >50 >50 CNE5CRF01_AE >50 >50 >50 BJOX009000.02.4 CRF01_AE >50 >50 3.626BJOX015000.11.5 CRF01_AE(T/F) >50 >50 >50 BJOX010000.06.2CRF01_AE(T/F) >50 >50 >50 BJOX025000.01.1 CRF01_AE(T/F) >50 >50 >50BJOX028000.10.3 CRF01_AE(T/F) >50 >50 >50 X1193_c1 G 0.144 0.083 0.045P04S2_c2_11 G 0.022 0.012 0.020 X1254_c3 G 0.121 0.089 0.056 X2088_c9 G0.002 0.003 0.011 X2131_C1_85 G 0.019 0.016 0.015 P1981_C5_3 G 0.0050.005 0.004 X1632_S2_B10 G >50 >50 >50 3016.v5.c45 D >50 >50 >50A07412M1.vrc12 D 0.008 <0.001 0.001 231965.c01 D >50 >50 >50 231966.c02D >50 >50 >50 191821_E6_1 D(T/F) >50 >50 >50 3817.v2.c59 CD 8.1473.148 >50 6480.v4.c25 CD 0.010 0.009 0.017 6952.v1.c20 CD 0.044 0.0370.085 6811.v7.c18 CD 0.001 0.002 0.004 89-F1_2_25 CD >50 >50 >503301.v1.c24 AC 0.016 0.013 0.014 6041.v3.c23 AC >50 >50 >50 6540.v4.c1AC >50 >50 >50 6545.v4.c1 AC >50 >50 >50 0815.v3.c3 ACD 0.061 0.0300.022 3103.v3.c10 ACD 0.053 0.037 0.042 Numbers indicate antibody lgGconcentrations in μg/ml to reach the IC₅₀ in the TZM-bl neutralizationassay. IC50 values indicate increasing neutralizationsensitivity. >indicates that the IC₅₀ for a given virus was not reachedat the concentration tested.

TABLE 6 In vitro TZM-bl neutralization assay on the extended panel -IC80 Virus ID Clade 10-996 10-1074 PGT121 6535.3 B 0.046 0.026 0.021QH0692.42 B 1.854 0.929 8.545 SC422661.8 B 0.627 0.418 0.460 PVO.4 B0.952 0.360 0.945 TR0.11 B 0.081 0.057 0.051 AC10.0.29 B 0.250 0.1100.169 RHPA4259.7 B 0.163 0.118 0.054 THRO4156.18 B >50 >50 >50REJO4541.67 B >50 >50 >50 TRJO4551.58 B 7.269 0.634 35.291 WITO4160.33 B6.484 2.112 6.007 CAAN5342.A2 B 0.079 0.036 0.051 YU-2 B 0.891 0.3980.356 WEAU_d15_410_787 B (T/F) 0.422 0.375 0.295 1006_11_C3_1601 B (T/F)0.019 0.013 0.023 1054_07_TC4_1499 B (T/F) 0.901 0.583 0.6961054_10_TA11_1826 B (T/F) 0.563 0.272 0.303 1012_11_TC21_3257 B (T/F)0.111 0.059 0.038 6240_08_TA5_4622 B (T/F) 0.348 0.306 0.5846244_13_B5_4576 B (T/F) 1.296 0.922 1.878 62357_14_D3_4589 B(T/F) >50 >50 45.559 SC05_8C11_2344 B (T/F) 0.174 0.123 0.275 Du156.12 C0.101 0.076 0.033 Du172.17 C 0.607 0.430 0.890 Du422.1 C 0.215 0.1660.131 ZM197M.PB7 C >50 >50 >50 ZM214M.PL15 C 3.251 2.367 3.150ZM233M.PB6 C 4.524 0.349 8.977 ZM249M.PL1 C >50 >50 >50 ZM53M.PB12C >50 >50 0.002 ZM109F.PB4 C >50 >50 >50 ZM135M.PL10a C 0.553 0.3675.885 CAP45.2.00.G3 C >50 >50 6.544 CAP210.2.00.E8 C >50 >50 >50HIV-001428-2.42 C 0.204 0.281 0.156 HIV-0013095-2.11 C >50 >50 >50HIV-16055-2.3 C >50 >50 4.290 HIV-16845-2.22 C 9.933 5.835 >50 Ce1086_B2C (T/F) >50 >50 0.006 Ce0393_C3 C (T/F) >50 >50 >50 Ce1176_A3 C (T/F)0.151 0.070 0.058 Ce2010_F5 C (T/F) >50 >50 >50 Ce0682_E4 C(T/F) >50 >50 >50 Ce1172_H1 C (T/F) 0.173 0.166 0.088 Ce2060_G9 C(T/F) >50 >50 >50 Ce703910054_2A2 C (T/F) >50 >50 >50 BF1266.431a C(T/F) >50 >50 >50 246F C1G C (T/F) 0.270 0.111 0.287 249M B10 C(T/F) >50 >50 >50 2M247v1(Rev-) C (T/F) 0.252 0.186 0.1267030102001E5(Rev-) C (T/F) 0.044 0.021 0.043 1394C9G1(Rev-1) C (T/F)0.328 0.191 3.372 Ce704809221_1B3 C (T/F) 1.208 0.696 0.492 CNE19BC >50 >50 0.189 CNE20 BC <0.001 0.005 0.008 CNE21 BC 0.255 0.181 0.061CNE17 BC 24.701 13.297 >50 CNE30 BC 1.989 1.200 0.559 CNE52 BC 43.83413.147 32.935 CNE53 BC 0.233 0.141 0.200 CNE58 BC 2.220 0.968 >50MS208.A1 A >50 >50 >50 Q23.17 A 0.030 0.021 0.031 Q451.e2 A >50 >50 >50Q769.d22 A >50 >50 >50 Q259.d2.17 A >50 >50 >50 Q842.d12 A >50 >50 0.0743415.v1.c1 A >50 >50 >50 3365.v2.c2 A 1.380 0.450 7.353 0260.v5.c36 A0.436 0.160 0.152 191955_A11 A (T/F) >50 >50 >50 191084 B7-19 A (T/F)0.144 0.128 0.128 9004SS_A3_4 A (T/F) 0.050 0.030 0.026 T257-31CRF02_AG >50 >50 >50 928-28 CRF02_AG 7.151 4.696 >50 263-8 CRF02_AG >506.527 24.576 T250-4 CRF02_AG 0.005 0.005 0.011 T251-18 CRF02_AG 7.3997.395 >50 T278-50 CRF02_AG >50 18.276 >50 T255-34 CRF02_AG >50 >50 >50211-9 CRF02_AG 3.848 0.425 8.840 235-47 CRF02_AG 0.381 0.163 1.676620345.c01 CRF01_AE >50 >50 >50 CNE8 CRF01_AE >50 >50 >50 C1080.c03CRF01_AE >50 >50 >50 R2184.c04 CRF01_AE >50 >50 >50 R1166.c01CRF01_AE >50 >50 >50 C2101.c01 CRF01_AE >50 >50 >50 C3347.c11CRF01_AE >50 >50 >50 C4118.c09 CRF01_AE >50 >50 >50 CNE5CRF01_AE >50 >50 >50 BJOX009000.02.4 CRF01_AE >50 >50 37.289BJOX015000.11.5 CRF01_AE(T/F) >50 >50 >50 BJOX010000.06.2CRF01_AE(T/F) >50 >50 >50 BJOX025000.01.1 CRF01_AE(T/F) >50 >50 >50BJOX028000.10.3 CRF01_AE(T/F) >50 >50 >50 X1193_c1 G 0.482 0.475 0.202P0402_c2_11 G 0.065 0.039 0.056 X1254_c3 G 0.420 0.297 0.199 X2088_c9 G0.014 0.014 0.029 X2131_C1_B5 G 0.085 0.064 0.058 P1981_C5_3 G 0.0180.017 0.015 X1632_S2_B10 G >50 >50 >50 3016.v5.c45 D >50 >50 >50A07412M1.vrc12 D 0.070 0.048 0.406 231965.c01 D >50 >50 >50 231966.c02D >50 >50 >50 191821_E6_1 D(T/F) >50 >50 >50 3817.v2.c59 CD 34.61914.880 >50 6480.v4.c25 CD 0.049 0.041 0.079 6952.v1.c20 CD 0.188 0.1380.605 6811.v7.c.18 CD 0.011 0.010 0.017 89-F1_2_25 CD >50 >50 >503301.v1.c24 AC 0.054 0.042 0.043 6041.v3.c23 AC >50 >50 >50 6540.v4.c1AC >50 >50 >50 6545.v4.c1 AC >50 >50 >50 0815.v3.c3 ACD 0.251 0.1380.105 3103.v3.c10 ACD 0.150 0.101 0.110 Numbers indicate antibody lgGconcentrations in μg/ml to reach the IC₈₀ in the TZM-bl enuralizationassay. IC₈₀ values indicate neutralization sensitivity. >indicatestahtthe IC₈₀ for a given virus was not reached at the concentrationtested.

TABLE 8 In vitro PBMC-based neutralization assay Virus ID 3BNC55 3BNC603BNC117 3BNC134 1NC9 45-46 3BNC195 12A12 4E10 b12 CONTEM- P035.6.EA1.918 0.023 <0.0032 0.034 0.451 0.037 >50 0.110 1.043 11.865 PORARYP035.6.H11 0.550 0.029 0.022 0.239 0.031 0.053 >50 0.130 2.941 10.758HISTOR- P035.6.D10 >50 >50 0.019 >50 0.248 <0.0032 >50 <0.00320.792 >12.5 ICAL P151.37.C7 0.084 0.038 0.053 0.128 0.018 0.016 0.1330.402 2.941 1.882 P151.37.F1 1.297 0.125 0.161 1.633 1.288 0.203 0.13618.043 0.860 >12.5 P151.37.F10 3.770 0.311 0.249 4.661 >50 0.375 >5018.883 9.314 0.452 P153.10.2.A9 15.804 0.763 0.861 17.208 46.735 0.0690.201 >50 12.269 7.988 P153.10.2.D8 20.568 1.020 0.206 23.124 >500.030 >50 2.478 0.861 >12.5 P153.10.2.E10 1.226 0.546 0.211 2.105 >500.108 >50 10.686 0.792 9.212 P186.12.1.D10 0.061 0.061 0.105 >50 0.0750.165 0.091 7.288 >25 9.333 P186.12.1.F4 >50 >50 >50 >50 >50 >50 >50 >504.903 11.075 P186.12.1.G2 1.890 <0.0032 <0.0032 >50 0.046 0.017 >500.068 15.711 10.763 P195.31.A6 0.032 0.138 <0.0032 0.171 0.228 0.0520.099 0.206 2.934 0.498 P195.31.A10 0.252 0.024 0.025 >50 0.953 0.0500.071 >50 1.248 9.896 P195.31F11 0.569 0.084 0.137 >50 >50 0.076 >5033.131 2.301 >12.5 P019.1.D2 0.949 0.164 0.027 >50 >50 0.057 >5014.612 >25 0.432 P019.1.D8 3.122 0.738 0.635 >50 >50 5.767 >50 >502.443 >12.5 P019.1.G7 6.034 3.636 4.570 >50 >50 6.962 >50 20.0921.073 >12.5 P175.10.D7 2.539 0.936 3.588 >50 42.695 17.723 >50 35.8051.187 1.369 P175.10.D12 0.708 0.410 0.067 >50 0.317 0.175 1.233 10.6480.953 >12.5 P175.10.G10 2.508 0.563 0.621 >50 33.008 8.364 22.413 18.5541.147 >12.5 P013.18.A9 0.125 0.102 0.079 >50 0.198 0.018 0.426 0.2050.786 >12.5 P154.44.C8 >50 0.683 1.042 >50 >50 16.4400.925 >50 >25 >12.5 P154.44.G8 >50 1.220 2.172 >50 >50 25.616 1.969 >504.450 >12.5 P183.50.2.H3 2.197 0.116 0.1212 12.049 4.079 0.161 >50 0.4763.890 5.923 P183.3.2.B9 0.076 0.025 <0.0032 0.053 0.326 0.142 >50 0.4721.722 1.230 P001.35.F5 0.418 0.046 0.047 8.133 0.329 0.133 >500.197 >0.39 >12.5 P001.35.H4 2.180 0.333 1.919 >50 >50 6.118 >50 >500.830 >12.5 P002.39.CB 15.016 0.117 0.172 2.192 12.154 0.546 >50 >501.955 >12.5 P002.39.FB >50 28.891 >50 13.090 >50 0.404 >50 >500.864 >12.5 P002.39.H10 1.472 0.785 0.726 14.160 1.147 0.017 >50 >501.530 >12.5 P034.6.D6 37.003 0.161 0.126 11.339 1.137 0.235 >50 0.8021.164 11.122 P034.6.G10 48.877 0.348 0.237 22.481 1.428 0.017 >50 0.1860.820 8.598 P034.6.H5 >50 0.417 0.267 20.730 0.820 0.245 >50 0.8660.391 >12.5 P101.20.1.F1 >50 >50 >50 >50 >50 0.565 >50 0.634 3.999 >12.5P101.20.1.HB >50 >50 >50 >50 >50 0.090 >50 0.474 1.902 >12.5P127.46.A6 >50 >50 >50 >50 >50 0.386 >50 0.666 1.211 6.780 P127.46.D11.242 0.024 0.043 5.403 2.558 0.169 >50 0.227 1.092 >12.5 P127.46.D21.125 0.173 0.221 >50 2.231 0.279 >50 0.494 1.613 <0.195 P174.28.E112.399 0.483 0.716 >50 13.061 0.894 >50 2.104 2.113 8.149 P177.25.1.G90.980 0.191 0.189 >50 1.826 0.261 >50 0.130 1.665 >12.5 P177.25.2.B41.179 0.080 0.041 23.609 0.384 0.150 >50 0.028 3.729 >12.5P177.25.2.D1 >50 1.949 1.359 >50 46.825 11.454 >50 7.681 1.140 7.770P180.14.A6 1.389 0.098 0.058 0.017 >50 0.024 >50 0.022 1.162 >12.5P180.14.G6 45.246 0.116 0.122 2.449 13.361 0.169 0.220 0.093 4.009 >12.5P180.14.G7 23.444 0.052 0.035 0.022 1.450 0.024 0.703 0.729 15.759 >12.5P197.25.1.D2 >50 0.285 0.194 1.480 1.137 0.016 0.028 0.072 1.492 >12.5P197.25.1.D7 >50 0.782 0.019 0.052 7.058 0.051 0.016 2.601 0.948 >12.5P197.25.1.H1 >50 0.017 0.019 <0.0032 <0.0032 0.029 <0.0032 0.7542.515 >12.5 P405.18.D3 0.068 <0.0032 0.029 0.095 0.048 0.022 0.022 0.0310.924 0.471 P405.18.F10 0.936 0.063 0.084 5.646 0.432 0.094 0.126 0.4691.350 0.716 P405.18.H5 4.726 0.219 0.782 1.100 19.220 0.027 0.450 43.6841.328 0.497 P405.19.A8 0.291 0.021 <0.0032 0.116 0.278 0.059 0.110 0.0342.012 1.116 P405.19.B12 0.889 0.057 0.098 0.264 1.103 0.033 0.233 0.1570.807 0.656 P405.19.F11 0.689 0.018 0.109 2.892 2.131 0.037 >50 5.2790.818 0.413 1140.6F5 0.328 0.029 <0.0032 5.219 6.915 0.284 >50 13.91721.480 >25 1170.6G9 0.748 0.116 0.114 4.222 0.096 0.147 >50 0.220 6.7103.270 P116.2 5.406 0.493 0.422 40.937 5.647 0.142 >50 17.250 16.58017.370 P116.3.F6 22.297 0.235 0.255 0.495 0.728 0.158 >50 15.152 9.52013.540 P116.3.G9 1.054 0.071 0.018 0.435 3.157 1.385 0.540 24.689 5.7500.650 P116.4.11 2.594 0.149 0.353 17.822 6.646 0.703 0.329 26.982 13.7903.120 1234.3A9 2.623 0.226 0.032 15.504 3.944 0.815 0.062 40.94024.600 >25 1234.3D9 0.563 0.102 0.057 4.784 0.539 0.087 0.178 48.77915.780 >25 658.8A6 4.860 0.355 0.386 >50 2.057 0.379 0.19631.416 >25 >25 658.8D2 2.832 0.264 0.201 40.617 1.651 0.347 0.1973.291 >25 2.000 658.8F8 <0.0032 <0.0032 <0.0032 >0.0032 0.994 0.142 >500.018 >25 >25 526.17-2C11 <0.0032 0.032 0.040 0.028 0.048 0.123 >500.049 ND ND 526.17-2G1 0.429 0.141 0.025 <0.0032 5.002 0.962 >50 0.353ND ND 526.17-2G3 2.825 0.170 0.120 4.692 0.687 0.110 >50 0.928 ND ND424.9F4 0.065 0.091 0.016 0.589 2.534 0.508 >50 2.757 ND ND 424.9H117.101 1.386 1.114 19.990 4.508 0.590 >50 4.982 ND ND 139.19A6 >50 1.0591.091 3.132 >50 0.090 >50 0.520 ND ND 139.19.C10 >50 0.118 0.0895.745 >50 0.019 >50 0.093 ND ND 139.19.F2 >50 0.241 0.226 0.755 11.1250.038 >50 0.525 ND ND 208.9.C6 17.496 0.375 0.587 25.217 1.510 0.5870.123 >50 ND ND 208.9.F12 5.263 0.265 0.314 11.871 4.460 0.414 0.234 >50ND ND 208.9.G10 6.842 0.151 0.351 2.896 2.099 0.832 0.093 >50 ND ND1031.12.6C4 0.701 0.024 0.089 0.321 9.717 0.057 >50 >50 17.730 1.1801031.12.7D5 0.140 <0.0032 <0.0032 0.023 19.210 0.231 >50 >50 13.7700.960 1031.12.9D9 <0.0032 <0.0032 <0.0032 <0.0032 0.071 0.030 >50 >5018.220 3.540 1.7.1A7 0.280 0.116 0.189 >50 6.800 0.176 0.0610.743 >25 >25 1.7.1D2 10.695 0.939 0.998 >50 >50 0.669 0.062 10.213 >2511.920 1.7.1G10 >50 0.199 0.185 0.871 1.506 0.745 0.266 0.307 >25 >25233.7.1B2 >50 0.100 0.212 >50 <0.0032 0.158 <0.0032 0.057 15.000 >25233.7.1C3 >50 0.973 1.771 >50 >50 >50 0.041 0.132 10.700 8.030233.7.1C11 >50 0.241 0.226 0.755 11.125 0.036 >50 0.525 13.370 18.480458.5.12B1 17.496 0.375 0.587 25.217 1.510 0.587 0.123 >50 0.880 9.990458.5.12E1 5.263 0.265 0.314 11.871 4.460 0.414 0.234 >50 2.110 1.850458.5.12G9 6.84 0.151 0.351 2.896 2.099 0.832 0.093 >50 9.440 4.910172.7C6 0.701 0.024 0.089 0.321 9.717 0.057 >50 >50 ND ND 172.7F11 0.1400.001 0.001 0.023 19.210 0.231 >50 >50 ND ND 172.7G5 0.001 0.001 0.0010.001 0.071 0.030 >50 >50 ND ND 1161.9G11 0.260 0.116 0.189 >50 6.8000.176 0.061 0.743 ND ND 1161.9C1 10.695 0.939 0.998 >50 >50 0.669 0.06210.213 ND ND 537.8.A11 >50 0.199 0.185 0.871 1.508 0.745 0.268 0.307 NDND 537.8.E6 >50 0.100 0.212 >50 0.001 0.158 0.001 0.057 ND ND537.8.E10 >50 0.973 1.771 >50 >50 >50 0.041 0.132 ND ND Virus ID 2G122F5 PG9 PG16 VRC01 45-46 54W PGT121 10-1074 CONTEM- P035.6.EA <0.393.480 >1 >1 <0.078 0.032 15.471 0.059 PORARY P035.6.H11 <0.392.115 >1 >1 0.160 0.018 0.667 0.413 HISTOR- P035.6.D10 <0.39 1.900 >1 >10.390 0.020 0.174 0.110 ICAL P151.37.C7 >25 3.350 <0.016 <0.016 <0.0780.109 >50 >50 P151.37.F1 >25 1.372 <0.016 <0.016 0.250 0.472 >50 >50P151.37.F10 >25 <0.39 <0.016 <0.016 1.160 0.172 >50 >50 P153.10.2.A90.825 0.918 >1 0.431 >5 34.912 42.623 1.466 P153.10.2.D8 3.562 24.193 >10.853 0.890 0.325 1.603 0.620 P153.10.2.E10 <0.39 22.382 >1<0.016 >5 >50 >50 >50 P186.12.1.D10 >25 2.492 >1 >1 >5 22.865 0.0210.212 P186.12.1.F4 >25 15.277 >1 >1 3.320 15.929 0.026 0.094P186.12.1.G2 21.986 3.181 >1 >1 >5 16.385 0.018 0.108 P195.31.A6 >253.884 >1 >1 0.890 >50 >50 >50 P195.31.A10 >25 <0.39 >1 >14.800 >50 >50 >50 P195.31F11 >25 2.306 >1 >1 1.930 >50 >50 >50 P019.1.D2<0.39 <0.39 <0.016 <0.016 0.220 0.051 >50 0.191 P019.1.D8 9.371 <0.39<0.016 <0.016 <0.078 0.043 0.025 0.528 P019.1.G7 6.764 <0.39 0.050 0.034<0.078 <0.0032 >50 0.318 P175.10.D7 <0.39 >25 >1 0.110 1.100 0.022 0.0680.097 P175.10.D12 <0.39 >25 >1 0.150 0.170 0.032 0.085 0.241 P175.10.G102.183 1.300 0.720 0.032 1.660 0.017 0.069 0.220 P013.18.A9 >252.763 >1 >1 >5 0.083 5.844 >50 P154.44.C8 >25 >25 <0.0160.600 >5 >50 >50 >50 P154.44.G8 >25 >25 <0.016 >1 0.790 0.536 1.9960.521 P183.50.2.H3 6.688 1.527 >1 >1 1.031 0.195 2.106 0.185 P183.3.2.B92.132 1.073 >1 >1 0.340 0.056 5.073 0.266 P001.35.F5 >25 >25 <0.016<0.016 >5 0.946 1.873 0.046 P001.35.H4 >25 >25 <0.016 <0.016 4.300 0.0512.648 0.052 P002.39.CB 2.216 >25 <0.016 <0.016 <0.078 0.450 1.129 0.024P002.39.FB <0.39 17.778 <0.016 <0.016 1.610 0.625 22.125 0.049P002.39.H10 <0.39 1.500 <0.016 0.048 3.030 0.917 1.985 0.042 P034.6.D60.844 0.873 <0.016 0.122 <0.078 0.859 0.027 <0.0032 P034.6.G10 <0.391.257 >1 >1 >5 >50 >50 0.028 P034.6.H5 >25 1.163 0.017 0.021 0.310 0.1270.037 0.018 P101.20.1.F1 >25 2.721 >1 >1 2.420 0.676 <0.0032 <0.0032P101.20.1.HB >25 1.719 >1 >1 1.470 0.861 <0.0032 <0.0032 P127.46.A62.345 1.671 0.079 <0.016 <0.078 0.143 >50 >50 P127.46.D1 <0.39 0.9210.101 0.150 0.230 0.203 >50 >50 P127.46.D2 >25 1.126 0.378 <0.016 <0.0780.023 >50 >50 P174.28.E11 0.974 1.674 0.023 <0.016 3.910 1.863 0.0850.050 P177.25.1.G9 >25 5.854 >1 >1 <0.078 0.325 0.128 0.034P177.25.2.B4 >25 1.241 >1 >1 0.450 0.530 0.029 0.023 P177.25.2.D1 >251.232 >1 >1 3.630 0.066 0.017 0.018 P180.14.A6 17.668 >25 0.038 <0.0161.000 2.094 0.139 0.028 P180.14.G6 2.988 >25 <0.016 <0.016 2.380 0.3060.174 0.020 P180.14.G7 2.400 >25 <0.016 <0.016 1.240 1.012 0.028 0.038P197.25.1.D2 >12.5 1.224 <0.016 <0.016 0.090 0.025 14.287 0.095P197.25.1.D7 1.232 1.291 <0.016 <0.016 <0.078 0.052 20.337 0.057P197.25.1.H1 1.079 2.088 <0.016 <0.016 <0.078 0.072 23.417 0.092P405.18.D3 <0.39 1.137 >1 >1 >5 0.254 0.071 0.018 P405.18.F10 <0.390.804 ND ND >5 >50 5.680 >50 P405.18.H5 0.986 0.815 0.141 <0.016 >5 >504.760 0.187 P405.19.A8 0.814 0.878 >1 0.044 0.470 0.031 0.917 <0.0032P405.19.B12 0.986 1.029 0.190 <0.016 >5 <0.0032 <0.0032 <0.0032P405.19.F11 <0.39 3.630 0.190 0.034 >5 ND ND ND 1140.6F5 >25 3.790<0.016 <0.016 <0.078 <0.0032 0.017 0.019 1170.6G9 >25 1.520 <0.016<0.016 <0.078 0.019 0.112 0.024 P116.2 >25 4.170 <0.016 <0.016 <0.078<0.0032 0.017 <0.0032 P116.3.F6 >25 3.080 <0.016 <0.016 <0.078 0.0200.019 <0.0032 P116.3.G9 >25 3.460 <0.016 <0.016 <0.078 <0.0032 0.033<0.0032 P116.4.11 >25 7.860 <0.016 <0.016 <0.078 <0.0032 <0.0032 <0.00321234.3A9 >25 >25 <0.016 <0.016 <0.078 <0.0032 0.022 <0.0032 1234.3D9 >258.550 <0.016 <0.016 <0.078 0.028 0.022 <0.0032 658.8A6 >25 >25 0.1420.032 <0.078 <0.0032 0.151 0.049 658.8D2 >25 17.210 0.135 <0.016 <0.078<0.0032 0.051 0.029 658.8F8 >25 >25 0.039 <0.016 <0.078 <0.0032 0.0260.017 526.17-2C11 ND ND <0.016 <0.016 0.199 0.046 <0.0032 <0.0032526.17-2G1 ND ND 0.059 <0.016 0.840 0.074 <0.0032 0.018 526.17-2G3 ND ND<0.016 <0.016 0.354 <0.0032 0.087 <0.0032 424.9F4 ND ND <0.016 <0.0160.310 0.028 0.032 0.019 424.9H1 ND ND 0.039 <0.016 >5 0.369 3.731 1.087139.19A6 ND ND <0.016 <0.016 0.557 0.127 0.068 0.017 139.19.C10 ND ND<0.016 <0.016 1.549 0.688 0.118 0.039 139.19.F2 ND ND <0.016 <0.016 >50.669 0.106 0.039 208.9.C6 ND ND 0.063 0.248 0.303 0.100 0.125 0.137208.9.F12 ND ND >1 >1 0.714 0.128 0.086 0.151 208.9.G10 ND ND 0.882 >10.289 0.083 <0.0032 0.023 1031.12.6C4 >25 6.280 >1 >1 0.160 0.040<0.0032 <0.0032 1031.12.7D5 >25 8.500 >1 >1 0.120 0.072 <0.0032 <0.00321031.12.9D9 >25 10.770 >1 >1 0.200 0.102 <0.0032 <0.0032 1.7.1A7 >25 >25<0.016 <0.016 0.640 ND ND ND 1.7.1D2 >25 >25 <0.016 <0.016 0.700 0.3000.045 0.048 1.7.1G10 >25 >25 <0.016 <0.016 <0.078 0.282 0.017 <0.0032233.7.1B2 0.140 5.030 0.019 <0.016 0.160 0.019 0.030 <0.0032 233.7.1C30.330 5.900 0.526 >1 <0.078 0.027 <0.0032 <0.0032 233.7.1C11 0.8705.460 >1 0.370 0.090 0.024 0.023 0.038 458.5.12B1 >25 3.820 0.028 0.3111.370 0.035 0.018 0.018 458.5.12E1 22.620 1.660 <0.016 <0.016 <0.0780.072 0.201 0.020 458.5.12G9 >25 2.350 >1 0.039 0.100 0.223 0.022 0.023172.7C6 ND ND 0.020 <0.016 0.431 0.019 0.025 0.027 172.7F11 ND ND 0.017<0.016 0.424 0.021 0.020 0.025 172.7G5 ND ND 0.048 <0.016 <0.078 <0.00320.035 <0.0032 1161.9G11 ND ND >1 >1 0.378 0.021 <0.0032 0.020 1161.9C1ND ND >1 >1 >5 0.618 0.065 0.051 537.8.A11 ND ND >1 >1 3.459 0.270 0.0310.021 537.8.E6 ND ND >1 >1 0.331 0.065 0.086 0.027 537.8.E10 ND ND >1 >12.071 0.174 0.025 0.017 Numbers indicate antibody lgG concentrations inμg/ml to reach the IC₅₀ in the PBMC-based neutralization assay. IC₅₀values indicate an increasing neutralization sensitivity. >indicatesthat the IC₅₀ for a given virus was not reached at the concetrationtested. ND, not determined.

TABLE 9 Data collection and refinement statistics (molecularreplacement) PGT121 Fab “unliganded” 10-1074 Fab GL Fab PGT121 Fab“liganded” Data collection Space group P2₁2₁2₁ P2₁ P2₁ P2₁2₁2₁ Celldimensions a, b, c (Å) 56.75, 74.67, 114.917 61.38, 40.26, 84.46 54.93,344.74, 55.23 67.79, 67.79, 94.11 α, β, γ (°) 90.00, 90.00, 90.00 90.00,95.39, 90.00 90.00, 91.95, 90.00 90.00, 90.00, 90.00 Resolution (Å)2.78-35.5 (2.78-2.93) 1.80-36.31 (1.80-1.91)  2.42-38.60 (2.42-2.55) 2.33-38.66 (2.33-2.47)  R_(merge) 0.099 (0.293) 0.075 (0.558) 0.072(0.482) 0.161 (0.603) I/σ_(i) 8.8 (3.1) 8.7 (1.8) 11.0 (1.9)  8.7 (2.9)Completeness (%) 96.7 (84.8) 93.49 (98.0) 95.5 (80.1) 92.2 (98.9)Redundancy 3.2 (2.7) 2.7 (2.8) 3.1 (2.6) 5.3 (5.8) Refinement Resolution(Å) 3.0 1.9 2.42 2.4 No. reflections 10,076 31,363 74,237 16,831R_(work)/R_(tree) 0.216/0.264 0.187/0.223 0.194/0.237 0.201/0.249 No.atoms Protein 3,276 3,346 12,881 3,127 Ligand/ion 0 0 0 129 Water 0 300527 203 B-factors Protein 32.78 29.17 44.67 31.48 Ligand/ion — — — 45.1Water — 37.37 40.27 36.78 R.m.s. deviations Bond lengths (Å) 0.005 0.0070.005 0.006 Bond angles (°) 0.971 1.234 0.951 0.949 *Data for eachstructure were acquired from a single crystal. *Values in parenthesesare for the highest-resolution shell.

TABLE 10 RMSD values for Cα alignments of Fabs Fab1/Fab2 RMSD_(VH) (Å) #residues RMSD_(VL) (Å) # residues RMSD_(VH+VL) (Å) # residuesPGT121/PGT128 1.159 116/130 1.63 95/100 1.462 207/235 PGT121/PGT145 2.93124/130 1.91 94/105 1.75 206/235 PGT121/10-1074 0.74 128/130 1.2102/105  1.26 226/235 PGT121/GL 1.33 129/130 1.37 94/105 1.6 225/23510-1074/GL 1.38 130/130 1.35 92/105 1.39 220/235PGT121/PGT121_(liganded) 0.79 125/128 0.5 100/100  0.78 225/228

TABLE 11 Contacts between PGT121 Fab and bound glycan Glycan atomProtein atom Water Distance (Å) GlcNAc⁶-O3 Asn⁵⁸-Nδ2 2.91 GlcNAc⁶-O7Asn⁵⁸-Oδ1 2.94 GlcNAc⁶-O6 H₂O⁴⁷¹ 3.15 GlcNAc⁶-O4 H₂O⁴⁷⁷ 3.05 GlcNAc⁶-O3H₂O⁴⁸¹ 2.94 Man¹-O4 H₂O⁴¹⁰ 3.02 Man¹-O4 H₂O⁴²⁰ 2.66 Man¹-O3 H₂O⁴¹⁰ 3.35Man¹-O2 H₂O⁴⁷⁷ 3.14 Man¹-O5 H₂O⁴⁷⁷ 2.62 Man²-O6 Thr¹⁰⁰-Oγ1 3.34 Man²-O2H₂O⁴¹⁰ 3.41 Man²-O5 H₂O⁴⁴⁶ 2.95 Man²-O6 H₂O⁴⁴⁶ 3.26 GlcNAc⁷-N2 Tyr³³-OH2.72 GlcNAc⁷-O5 H₂O⁴¹⁰ 3.38 GlcNAc⁷-O7 H₂O⁴¹¹ 3.00 GlcNAc⁷-O3 His⁹⁷-Nδ23.60* GlcNAc⁷-O7 His⁹⁷-Nε2 3.70* Gal⁸-O3 Lys⁵³-Nζ 2.97 Gal⁸-O4 H₂O⁴⁸⁰3.47 Gal⁸-O4 H₂O⁴³⁵ 2.76 Gal⁸-O5 H₂O⁴³⁵ 3.17 Sia¹⁰-O8 Asp³¹-O 2.72Sia¹⁰-O10 His⁹⁷-N 3.18 Sia¹⁰-O10 His⁹⁷-O 3.19 Sia¹⁰-O9 H₂O⁴⁸⁰ 3.19Sia¹⁰-O8 Ser³²-Oγ 3.70* Man³-O3 Asn⁵⁸-Oδ1 2.58 Man³-O6 H₂O⁴⁷⁷ 3.35GlcNAc⁴-O5 Thr⁵⁷-O 3.33 GlcNAc⁴⁻N2 H₂O⁴⁷⁹ 3.2 Fuc⁹-O2 H₂O⁴⁷¹ 2.57Asp³¹-O H₂O⁴³⁵ 3.09 Asp³¹-Oδ1 H₂O⁴⁸⁰ 3.32 Tyr⁵⁰-OH H₂O⁴⁸¹ 2.8 His⁵²-Nε2H₂O⁴³⁵ 3.16 Ser⁵⁴-Oγ H₂O⁴¹⁰ 3.2 Ser⁵⁴-O H₂O⁴²⁰ 3.16 Gly⁵⁵-O H₂O⁴⁷⁹ 2.85Asp⁵⁶-Oδ1 H₂O⁴⁸¹ 3.49 Asp⁵⁶-Oδ2 H₂O⁴⁴⁸ 3.07 Asn⁵⁸-Nδ2 H₂O⁴⁸¹ 3.15Arg⁹⁹-Nε H₂O⁴¹¹ 2.58 Thr¹⁰⁰¹-Oγ1 H₂O⁴¹¹ 2.94 Hydrogen bond criteria:bond distance <3.5 Å, O—H—O/N—H—O angle >90° *Contacts are close tohydrogen bond distance cutoff and are included as possible interactions

TABLE 12 In vitro neutralization activity of PGT121GM and 10-1074GMVirus ID Clade PGT121 PGT121GM 10-1074 10-1074GM Q842.d12 A0.074 >50 >50 >50 3365.v2.c2 A 7.353 >50 0.450 0.467 0260.v5.c36 A0.152 >50 0.160 0.618 YU.2 B 0.356 1.355 0.398 0.262 TRO.11 B 0.0510.258 0.057 0.049 TRJO4551.58 B 35.291 >50 0.634 0.721 QH0692.42 B8.545 >50 0.929 0.376 PVO.4 B 0.945 47.564 0.360 0.138 RHPA4259.7 B0.054 20.801 0.118 0.087 WITO4160.33 B 6.007 >50 2.112 0.4061054_07_TC4_1499 B (T/F) 0.696 >50 0.563 0.193 6244_13_B5_4576 B (T/F)1.878 46.680 0.922 0.394 62357_14_D3_4589 B (T/F) 45.559 >50 >50 40.782CNE19 BC 0.189 48.092 50 0.379 CNE17 BC >50 >50 13.297 4.816 CNE58BC >50 >50 0.968 1.158 CNE30 BC 0.559 8.401 1.200 1.045 CNE52 BC32.935 >50 13.147 6.664 ZM233M.PB6 C 8.977 >50 0.349 0.232 ZM53M.PB12 C0.002 >50 >50 >50 CAP45.2.00.G3 C 6.544 >50 >50 >50 HIV-16055-2.3 C4.290 >50 >50 >50 HIV-16845-2.22 C >50 >50 5.835 2.678 ZM214M.PL15 C3.150 >50 2.367 0.200 ZM135M.PL10a C 5.885 >50 0.367 0.184 Ce1086_B2 C(T/F) 0.006 >50 >50 >50 Ce1172_H1 C (T/F) 0.088 0.180 0.166 0.0541394C9G1(Rev-) C (T/F) 3.372 2.120 0.191 0.075 3817.v2.c59 CD >50 >5014.880 3.423 6952.v1.c20 CD 0.605 >50 0.138 0.134 BJOX009000.02.4CRF01_AE 37.289 >50 >50 >50 211-9 CRF02_AG 8.840 >50 0.425 0.976 928-28CRF02_AG >50 >50 4.696 3.121 T251-18 CRF02_AG >50 >50 7.395 3.459T278-50 CRF02_AG >50 >50 18.276 12.017 263-8 CRF02_AG 24.576 >50 6.5277.779 235-47 CRF02_AG 1.676 >50 0.163 0.069 A07412m1.vrv12 D 0.40616.947 0.048 0.044 X1193 c1 G 0.202 11.859 0.475 0.195 X1254_c3 G 0.1990.222 0.297 0.112 Numbers indicate antibody IgG concentrations in μg/mlto reach the IC₅₀ in the TZM-bl neutralization assay. IC₅₀ valuesindicate an increasing neutralization sensitivity. >indicates that theIC₅₀ for a given virus was not reached at the concentration tested.

TABLE 13 SHIV_(AD8EO) Abs conc Titer (TZM- (μg/ml) bl) Animal ID AbsDosage PROTECTED at Day 0 at Day 0 RHDEGF VRC01 50 mg/Kg Yes 586.9 1:162RHDEH3 No 711.0 1:176 RHDE1L 20 mg/Kg No 206.5 1:65  RHJBN No 188.11:68  RHKNX PGT121 20 mg/Kg Yes 267.9  1:2495 RHMK4 Yes 253.6  1:2773RHDE9J  5 mg/Kg Yes 55.7 1:563 RHPNR No 47.2 1:618 RHDCGI  1 mg/Kg Yes24.0 1:116 RHKNE Yes 19.7 1:55  RHK44 0.2 mg/Kg  No 1.8 <1:20  RHK49 No1.8 1:17  RHDEEM 10-1074 20 mg/Kg Yes 289.8  1:2004 RHKIL Yes 257.7 1:2075 RHME1  5 mg/Kg Yes 112.9 1:633 RHPNV Yes 117.5 1:384 RHPID  1mg/Kg No 19.9 1:56  RHDCHX No 24.8 1:53  RHPZE 3BNC117  5 mg/Kg Yes105.4 1:272 RHPM5 Yes 76.1 1:372 RHKMH  1 mg/Kg No 39.6 1:55  RHMJ5 No15.1 1:75  RHPLD 45-46m2 20 mg/Kg No 15.0 1:27  RHMA9 No 17.6 <1:20 RHMC6  5 mg/Kg No 2.3 <1:20  RHDE0CA No 2.2 <1:20  RHML1 DEN3 20 mg/KgNo ND <1:20  RHMAA No ND <1:20  SHIV_(DH12-V3AD8) Abs conc (μg/ml) Titer(TZM-bl) Animal ID Abs dosage PROTECTED at Day 0 at Day 0 RHDEJ3 VRC0130 mg/Kg Yes 395.8 1:52  RHKZ1 No 306.0 1:70  RHKZA PGT121 20 mg/Kg Yes215.1  1:13120 RHDECT Yes 200.7  1:13805 RHKTL Yes 282.7  1:12669 RHPZ9Yes 133.1  1:12055 RHK2Z  1 mg/Kg Yes 15.1 1:422 RHMT8 No 29.3 1:539RHDEEB 0.2 mg/Kg  Yes 3.1 1:159 RHDEP2 Yes 1.6 1:101 RHMFD 0.05 mg/Kg  No 1.0 <1:20  RHKIA No 1.3 <1:20  RHKIM 10-1074 20 mg/Kg Yes 290.3 1:1972 RHKWM Yes 173.3  1:2282 RHMJW  5 mg/Kg Yes 96.6 1:420 RHMJT Yes95.3 1:376 RHDENI  1 mg/Kg Yes 28.4 1:106 RHJHZ No 18.6 1:136 RHHE8 0.2mg/Kg  No 19.4 1:39  RHKCZ No 19.7 1:35  RHMFBA 3BNC117 20 mg/Kg Yes294.9 1:143 RHMER Yes 272.7 1:142 RHKIV  5 mg/Kg Yes 114.6 1:80  RHKPINo 133.1 1:90  RHDE9D  1 mg/Kg No 23.3 1:20  RHDEW7 Yes 29.6 1:18  RHMEV0.2 mg/Kg  No 3.9 <1:20  RHMF9 No 5.7 <1:20  RHKZMA 45-46m2  5 mg/Kg No2.1 ND RHKNP No 4.0 ND RHJII hu-IgG 100 mg/Kg  No ND ND RHJK1 No ND ND

TABLE 14 IC₅₀ in TZM-bl cells¹ Sample HIVIG Tier ID S321 C500 B520 G435T520b M263 M600c (μg/ml) Phenotype R5 SHIV_(DH12-V3AD8) 321 289 77 172168 429 134 132 2 R5 SHIV_(AD8-EO) 48 36 39 31 41 44 48 1768 2 X4SHIV_(DH12-CL7) 110 94 50 65 109 115 65 530 2 HIV-1_(CAAN5342.A2) 84 <2027 <20 <20 77 185 638 2 HIV-1_(MN.3) 13944 9152 822 8432 3968 43722 17091.81 1 ¹Values are the serum dilution at which relative luminescenceunits (RLUs) were reduced 50% compared to virus control wells (no testsample).

TABLE 15 SHIVAD8EO Endpoint neutralization Animal number Accumulatedvalue Protected titer in plasma Protected Infected Protected^(a)Infected^(b) Ratio % 2773 1 0 12 0 12/12 100%  2495 1 0 11 0 11/11 100% 2075 1 0 10 0 10/10 100%  2004 1 0 9 0 9/9 100%  633 1 0 8 0 8/8 100% 618 0 1 7 1 7/8 88% 563 1 0 7 1 7/8 88% 384 1 0 6 1 6/7 86% 372 1 0 5 15/6 83% 272 1 0 4 1 4/5 80% 176 0 1 3 2 3/5 60% 162 1 0 3 2 3/5 60% 1151 0 2 2 2/4  50%^(c) 75 0 1 1 3 1/4 25% 68 0 1 1 4 1/5 20% 65 0 1 1 51/6 17% 56 0 1 1 6 1/7 14% 55 1 0 1 6 1/7 14% 55 0 1 0 7 0/7  0% 53 0 10 8 0/8  0% 27 0 1 0 9 0/9  0% 20 0 1 0 10  0/10  0% 20 0 1 0 11  0/11 0% 20 0 1 0 12  0/12  0% 20 0 1 0 13  0/13  0% 17 0 1 0 14  0/14  0%^(a)Sum from the bottom. ^(b)Sum from the top. ^(c)Endpoint protectiontiter (50% protective titer) was calculated to be 1:115

TABLE 16 SHIVDH12-V3AD8 Endpoint neutralization Animal numberAccumulated value Protected titer in plasma Protected InfectedProtected^(a) Infected^(b) Ratio % 13805 1 0 16 0 16/16 100%  13120 1 015 0 15/15 100%  12669 1 0 14 0 14/14 100%  12055 1 0 13 0 13/13 100% 2282 1 0 12 0 12/12 100%  1972 1 0 11 0 11/11 100%  539 0 1 10 1 10/1191% 422 1 0 10 1 10/11 91% 420 1 0 9 1  8/10 90% 376 1 0 8 1 8/9 89% 1591 0 7 1 7/8 88% 143 1 0 6 1 6/7 86% 142 1 0 5 1 5/6 83% 136 0 1 4 2 4/667% 108 1 0 4 2 4/6 67% 101 1 0 3 2 3/5  60%^(c) 90 0 1 2 3 2/5  40%^(c)80 1 0 2 3 2/5 40% 70 0 1 1 4 1/5 20% 52 1 0 1 4 1/5 20% 39 0 1 0 5 0/5 0% 35 0 1 0 6 0/6  0% 20 0 1 0 7 0/7  0% 20 0 1 0 8 0/8  0% 20 0 1 0 90/9  0% 20 0 1 0 10  0/10  0% 20 0 1 0 11  0/11  0% 20 0 1 0 12  0/12 0% 20 0 1 0 13  0/13  0% ^(a)Sum from the bottom. ^(b)Sum from the top.^(c)Endpoint protection titer (50% protective titer) was calculated tobe 1:95.5

TABLE 17 Pre-infection Pre mAb Treatment Weeks CD4+ T Cells CD4+ T cellsViral Load Animal Post infection cells/μl cells/μl RNA Copies/mlClinical Status DBZ3 159 650 118 1.08E+04 Asymptomatic DC99A 159 623 1657.60E+03 Asymptomatic DBXE 163 1585 158 1.96E+05 Intermittent diarrheaDCF1 157 1203 105 1.44E+05 Intermittent diarrhea DCM8 163 608 431.59E+03 Intermittent diarrhea

TABLE 18 SIV Gag RNA SIV Gag DNA Treatment Copies per 10⁸ Copies per 10⁸Animal Time (Days) Cell Eq Cell Eq DBZ3 0 9,000 6,700 DBZ3 10 360 7,600DBZ3 20 2,400 14,000 DC99A 0 31,000 1,400 DC99A 14 18,000 5,600 DC99A 208,100 2,700 DBXE 0 470,000 71,000 DBXE 14 17,000 33,000 DBXE 17 11,40022,000 DCM8 0 110,000 8,600 DCM8 14 1,700 1,600 DCM8 20 22,000 6,800DCF1 0 240,000 15,400 DCF1 14 190,000 11,000 DCF1 20 1,100,000 14,000

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated herein in their entireties.

1. An isolated anti-HIV antibody, or antigen binding portion thereof,comprising at least one complementarity determining region (CDR) havinga sequence selected from the group consisting of SEQ ID NOs: 33-38. 2.The isolated anti-HIV antibody of claim 1, or antigen binding portionthereof, wherein the CDR comprises a sequence selected from the groupconsisting of SEQ ID NOs: 39-104.
 3. The isolated anti-HIV antibody ofclaim 1, or antigen binding portion thereof, comprising a heavy chainvariable region that comprises CDRH 1, CDRH 2, and CDRH 3, wherein theCDRH 1, CDRH 2 and CDRH 3 comprise the respective sequences of SEQ IDNOs: 33-35.
 4. The isolated anti-HIV antibody of claim 3, or antigenbinding portion thereof, wherein the CDRH 1, CDRH 2 and CDRH 3 comprisethe respective sequences of a CDRH set selected from the groupconsisting of SEQ ID NOs: 39-41, SEQ ID NOs: 45-47, SEQ ID NOs: 51-53,SEQ ID NOs: 57-59, SEQ ID NOs: 63-65, SEQ ID NOs: 69-71, SEQ ID NOs:75-77, SEQ ID NOs: 81-83, SEQ ID NOs: 87-89, SEQ ID NOs: 93-95, SEQ IDNOs: 99-101, and SEQ ID NOs: 131-133.
 5. The isolated anti-HIV antibodyof claim 1, or antigen binding portion thereof, comprising a light chainvariable region that comprises CDRL 1, CDRL 2 and CDRL 3, wherein theCDRL 1, CDRL 2 and CDRL 3 comprise the respective sequences of SEQ IDNOs: 36-38.
 6. The isolated anti-HIV antibody of claim 5, or antigenbinding portion thereof, wherein the CDRL 1, CDRL 2 and CDRL 3 comprisethe respective sequences of a CDRL set selected from the groupconsisting of SEQ ID NOs: 42-44, SEQ ID NOs: 48-50, SEQ ID NOs: 54-56,SEQ ID NOs: 60-62, SEQ ID NOs: 66-68, SEQ ID NOs: 72-74, SEQ ID NOs:78-80, SEQ ID NOs: 84-86, SEQ ID NOs: 90-92, SEQ ID NOs: 96-98, SEQ IDNOs: 102-104, and SEQ ID NOs: 134-136.
 7. The isolated anti-HIV antibodyof claim 1, or antigen binding portion thereof, comprising a heavy chainvariable region that comprises CDRH 1, CDRH 2, and CDRH 3, and a lightchain variable region that comprises CDRL 1, CDRL 2 and CDRL
 3. 8. Theisolated anti-HIV antibody of claim 7, or antigen binding portionthereof, wherein the CDRH 1, CDRH 2, CDRH 3, CDRL 1, CDRL 2 and CDRL 3comprise the respective sequences of a CDR set selected from the groupconsisting of SEQ ID NOs: 39-44, SEQ ID NOs: 45-50, SEQ ID NOs: 51-56,SEQ ID NOs: 57-62, SEQ ID NOs: 63-68, SEQ ID NOs: 69-74, SEQ ID NOs:75-79, SEQ ID NOs: 81-86, SEQ ID NOs: 87-92, SEQ ID NOs: 93-98, SEQ IDNOs: 99-104, and SEQ ID NOs: 131-136.
 9. The isolated anti-HIV antibodyof claim 1, or antigen binding portion thereof, comprising one or bothof (i) a heavy chain comprising the consensus amino acid sequence of SEQID NO: 1 and (ii) a light chain comprising the consensus amino acidsequence of SEQ ID NO:
 2. 10. The isolated anti-HIV antibody of claim 9,or antigen binding portion thereof, wherein the heavy chain comprises asequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, and 129 and the light chain comprises asequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, and
 130. 11. The isolated anti-HIV antibodyof claim 9, or antigen binding portion thereof, wherein the heavy chainand the light chain comprise the respective sequences of SEQ ID NOs:3-4, SEQ ID NOs: 5-6, SEQ ID NOs: 7-8, SEQ ID NOs: 9-10, SEQ ID NOs:11-12, SEQ ID NOs: 13-14, SEQ ID NOs: 15-16, SEQ ID NOs: 17-18, SEQ IDNOs: 19-20, SEQ ID NOs: 21-22, SEQ ID NOs: 23-24, and 129-130.
 12. Theisolated anti-HIV antibody of claim 1, or antigen binding portionthereof, wherein the antibody is one selected from the group consistingof 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130,10-1146, 10-1341, 10-1369, and 10-1074GM.
 13. The isolated anti-HIVantibody of anyone of claims 1-12, or antigen binding portion thereof,wherein the antibody is a human antibody, a humanized antibody, or achimeric antibody.
 14. An isolated nucleic acid comprising a sequenceencoding a CDR, a heavy chain variable region, or a light chain variableregion of the anti-HIV antibody of anyone of claims 1-13, or antigenbinding portion thereof.
 15. A vector comprising the nucleic acid ofclaim
 14. 16. A cultured cell comprising the vector of claim
 15. 17. Apharmaceutical composition comprising (i) at least one anti-HIV antibodyof anyone of claims 1-13, or antigen binding portion thereof, and (ii) apharmaceutically acceptable carrier.
 18. A method of preventing ortreating an HIV infection or an HIV-related disease comprising the stepsof: identifying a patient in need of such prevention or treatment, andadministering to said patient a first therapeutic agent comprising atherapeutically effective amount of at least one anti-HIV antibody ofanyone of claims 1-13, or antigen binding portion thereof.
 19. Themethod of claim 18, further comprising administering a secondtherapeutic agent.
 20. The method of claim 19, wherein the secondtherapeutic agent is an antiviral agent.
 21. A method for making ananti-HIV antibody or a fragment thereof, comprising obtaining thecultured cell of claim 16; culturing the cell in a medium underconditions permitting expression of a polypeptide encoded by the vectorand assembling of an antibody or fragment thereof, and purifying theantibody or fragment from the cultured cell or the medium of the cell.22. A kit comprising a pharmaceutically acceptable dose unit of apharmaceutically effective amount of at least one isolated anti-HIVantibody according to any of claims 1-13, and a pharmaceuticallyacceptable dose unit of a pharmaceutically effective amount of ananti-HIV agent, wherein the two pharmaceutically acceptable dose unitscan optionally take the form of a single pharmaceutically acceptabledose unit.
 23. The kit of claim 22, wherein the anti-HIV agent is oneselected from the group consisting of a non-nucleoside reversetranscriptase inhibitor, a protease inhibitor, a entry or fusioninhibitor, and an integrase inhibitor.
 24. A kit for the diagnosis,prognosis or monitoring the treatment of an HIV infection in a subjectcomprising one or more detection reagents which specifically bind toanti-HIV neutralizing antibodies in a biological sample from a subject.25. The kit of claim 24, further comprising reagents for performing PCR.26. The kit of claim 25, further comprising reagents for performing massspectrometry.